Particles, Compositions and Methods for Ophthalmic and/or Other Applications

ABSTRACT

This disclosure relates to particles, compositions, and methods that aid particle transport in mucus are provided. The particles, compositions, and methods may be used, in some instances, for ophthalmic and/or other applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under to U.S. ProvisionalPatent Application 62/395,984 filed Sep. 16, 2016, the entire contentsof which are incorporated by reference herein.

FIELD

The present disclosure generally relates to particles, compositions, andmethods that aid particle transport in mucus. The particles,compositions, and methods may be used in ophthalmic and/or otherapplications.

BACKGROUND

A mucus layer present at various points of entry into the body,including the eyes, nose, lungs, gastrointestinal tract, and femalereproductive tract, is naturally adhesive and serves to protect the bodyagainst pathogens, allergens, and debris by effectively trapping andquickly removing them via mucus turnover. For effective delivery oftherapeutic, diagnostic, or imaging particles via mucus membranes, theparticles must be able to readily penetrate the mucus layer to avoidmucus adhesion and rapid mucus clearance.

Particles (including microparticles and nanoparticles) that incorporatepharmaceutical agents are particularly useful for ophthalmicapplications. However, often it is difficult for administered particlesto be delivered to an eye tissue in effective amounts due to rapidclearance and/or other reasons. Accordingly, new methods andcompositions for administration (e.g., topical application or directinjection) of pharmaceutical agents to the eye would be beneficial.

SUMMARY

Disclosed herein are pharmaceutical compositions comprisingmucus-penetrating particles containing hydrocortisone(4-pregenen-11β-17-21-triol-3,20-dione) derivatives. In certainembodiments, the derivative is:

In some embodiments, the hydrocortisone derivative is(10R,11S,13S,17R)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl3-(phenylsulfonyl)propanoate (Compound 1),(10R,11S,13S,17R)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-ylfuran-2-carboxylate (Compound 2), or(10R,11S,13S,17R)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl2-(4-bromophenyl)acetate (Compound 3).

Some embodiments include a pharmaceutical composition suitable foradministration to an eye, comprising: a plurality of coated particles,comprising a core particle comprising a hydrocortisone derivativeselected from Compounds 1, 2, and 3; a mucus penetration-enhancingcoating comprising a surface-altering agent surrounding the coreparticle, wherein the surface-altering agent comprises: a) a triblockcopolymer comprising a hydrophilic block-hydrophobic block-hydrophilicblock configuration, wherein the hydrophobic block has a molecularweight of at least about 2 kDa, and the hydrophilic blocks constitute atleast about 15 wt % of the triblock copolymer, the hydrophobic blockassociates with the surface of the core particle, and the hydrophilicblock is present at the surface of the coated particle and renders thecoated particle hydrophilic, b) a synthetic polymer having pendanthydroxyl and ester groups in the backbone of the polymer, the polymerhaving a molecular weight of at least about 1 kDa and less than or equalto about 1000 kDa, wherein the polymer is at least about 30% hydrolyzedand less than about 95% hydrolyzed, or c) a polysorbate; wherein thesurface altering agent is present on the outer surface of the coreparticle at a density of at least 0.01 molecules/nm², wherein thesurface altering agent is present in the pharmaceutical composition inan amount of between about 0.001% to about 5% by weight; and anophthalmically acceptable carrier, additive, or diluent.

Some embodiments include a pharmaceutical composition suitable fortreating an ocular disorder by administration to an eye, comprising: aplurality of coated particles, comprising a core particle comprising ahydrocortisone derivative disclosed herein and a mucuspenetration-enhancing coating comprising a surface-altering agentsurrounding the core particle, wherein the surface-altering agentcomprises: a) a triblock copolymer comprising a hydrophilicblock-hydrophobic block-hydrophilic block configuration, wherein thehydrophobic block has a molecular weight of at least about 2 kDa, andthe hydrophilic blocks constitute at least about 15 wt % of the triblockcopolymer, b) a synthetic polymer having pendant hydroxyl groups on thebackbone of the polymer, the polymer having a molecular weight of atleast about 1 kDa and less than or equal to about 1000 kDa, wherein thepolymer is at least about 30% hydrolyzed and less than about 95%hydrolyzed, or c) a polysorbate, wherein the plurality of coatedparticles have an average smallest cross-sectional dimension of lessthan about 1 micron; and wherein the coating on the core particle ispresent in a sufficient amount to increase the concentration of thehydrocortisone derivative in a cornea or an aqueous humor afteradministration to the eye, compared to the concentration of thehydrocortisone derivative in the cornea or the aqueous humor whenadministered as a core particle without the coating.

Also provided herein are methods of treating, diagnosing, preventing, ormanaging an ocular condition in a subject, the method comprising:administering a pharmaceutical composition described herein, such as acomposition comprising a hydrocortisone derivative-containingmucus-penetrating particles to an eye of a subject and therebydelivering the hydrocortisone derivative to a tissue in the eye of thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a mucus-penetrating particle having acoating and a core according to one set of embodiments.

FIG. 2A depicts a histogram showing the ensemble averaged velocity<V_(mean)> in human cervicovaginal mucus (CVM) for 200 nm carboxylatedpolystyrene particles (PSCOO⁻; negative control), 200 nm PEGylatedpolystyrene particles (positive control), and nanoparticles (sample)made by milling and coated with different surface-altering agentsaccording to one set of embodiments. FIG. 2B is a plot showing therelative velocity <V_(mean)>_(rel) in CVM for nanoparticles made bymilling and coated with different surface-altering agents according toone set of embodiments.

FIGS. 3A-3D are histograms showing distribution of trajectory-meanvelocity V_(mean) in CVM within an ensemble of nanoparticles coated withthe surface-altering agents Pluronic® F127 (FIG. 3A), Pluronic® F87(FIG. 3B), Pluronic® F108 (FIG. 3C), and Kollidon 25 (FIG. 3D) accordingto one set of embodiments.

FIG. 4 is a plot showing <V_(mean)>_(rel) in CVM for nanoparticlescoated with different poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide) (PEO-PPO-PEO) Pluronic® triblock copolymers,mapped with respect to molecular weight of the PPO block and the PEOweight content (%), according to one set of embodiments.

FIG. 5A is a histogram showing the ensemble averaged velocity <V_(mean)>in human CVM for PSCOO⁻ particles coated with various poly(vinylalcohols) (PVAs) according to one set of embodiments. FIG. 5B is a plotshowing the relative velocity <V_(mean)>_(rel) in CVM for PSCOO⁻particles coated with various PVAs according to one set of embodiments.

FIG. 6 is a plot showing relative velocity <V_(mean)>_(rel) in CVM forPSCOO⁻ particles incubated with various PVAs mapped according to thePVA's molecular weight and degree of hydrolysis, according to one set ofembodiments. Each data point represents <V_(mean)>_(rel) for theparticles stabilized with a specific PVA.

FIGS. 7A-7B are plots showing bulk transport in CVM in vitro of PSCOO⁻nanoparticles coated with various PVAs in two different CVM samples,according to one set of embodiments. Negative controls are uncoated 200nm PSCOO⁻ particles; Positive controls are 200 nm PSCOO⁻ particlescoated with Pluronic® F127.

FIGS. 8A-8B are plots showing ensemble-average velocity <V_(mean)> (FIG.8A) and relative sample velocity <V_(mean)>_(rel) (FIG. 8B) forpoly(lactic acid) (PLA) nanoparticles (sample) prepared byemulsification with various PVAs as measured by multiple-particletracking in CVM, according to one set of embodiments.

FIGS. 9A-9B are plots showing ensemble-average velocity <V_(mean)> (FIG.9A) and relative sample velocity <V_(mean)>_(rel) (FIG. 9B) for pyrenenanoparticles (sample) and controls as measured by multiple-particletracking in CVM, according to one set of embodiments.

FIGS. 10A-10F are representative CVM velocity (V_(mean)) distributionhistograms for pyrene nanoparticles obtained with surface-alteringagents PVA2K75 (FIG. 10A), PVA9K80 (FIG. 10B), PVA31K98 (FIG. 10C),PVA85K99 (FIG. 10D), Kollidon 25 (FIG. 10E), and Kollicoat IR (FIG. 10F)(SAMPLE=pyrene nanoparticles, POSITIVE=200 nm PS-PEG5K, NEGATIVE=200 nmPS-COO); according to one set of embodiments.

FIG. 11 is a plot of relative velocity <V_(mean)>_(rel) for pyrenenanoparticles coated with PVA in CVM mapped according to the PVA'smolecular weight and degree of hydrolysis according to one set ofembodiments.

FIG. 12 is a bar graph showing the density of Pluronic® F127 on thesurface of fluticasone propionate and loteprednol etabonatemicroparticles, according to one set of embodiments.

FIG. 13 is a plot showing the mass transport through CVM for solidparticles having different core materials that are coated with eitherPluronic® F127 (MPP, mucus-penetrating particles) or sodium dodecylsulfate (CP, conventional particles, a negative control), according toone set of embodiments.

FIG. 14 depicts the X-ray powder diffraction (XRPD) pattern ofcrystalline form 2-A, according to one set of embodiments.

FIG. 15 depicts the XRPD pattern of crystalline form 3-A, according toone set of embodiments.

FIG. 16 depicts the XRPD pattern of crystalline form 3-B, according toone set of embodiments.

FIG. 17 depicts the XRPD pattern of crystalline form 1-B, according toone set of embodiments.

DETAILED DESCRIPTION

A pharmaceutical composition described herein (referred to herein as a“subject composition”) includes a drug-containing particle having amodification to a property of its surface. Although there are a numberof surface properties that may be modified, some embodiments relate tosurfaces that are modified to provide reduced adhesion to mucus orimproved penetration of the particles through physiological mucus, ascompared to unmodified drug-containing particles. Thus, disclosed hereinare subject compositions comprising mucus-penetrating particlescomprising a pharmaceutical composition coated with a mucuspenetration-enhancing surface-altering agent.

Particles having efficient transport through mucus barriers may bereferred to herein as mucus-penetrating particles (MPPs). The particlesmay more readily penetrate the mucus layer of a tissue to avoid orminimize mucus adhesion and/or rapid mucus clearance. Therefore, drugscontained in MPPs may be more effectively delivered to, and may beretained longer in, the target issue. As a result, the drugs containedin MPPs may be administered at a lower dose and/or less frequently thanformulations lacking MMPs to achieve similar or superior exposure.Moreover, the relatively low and/or infrequent dosage of the subjectcompositions may result in fewer or less severe side effects, and/orimproved patient compliance.

Non-limiting examples of mucosal tissues include oral (e.g., includingthe buccal and esophageal membranes and tonsil surface), ophthalmic,gastrointestinal (e.g., including stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., including nasal,pharyngeal, tracheal and bronchial membranes), and genital (e.g.,including vaginal, cervical and urethral membranes) tissues.

Examples of pharmaceutical applications that may benefit from theseproperties include including drug delivery, imaging, and diagnosticapplications. For example, a subject composition may be well-suited forophthalmic applications, and may be used for delivering pharmaceuticalagents to the front of the eye, middle of the eye, and/or the back ofthe eye. With respect to the front of the eye, MPPs may reduce dosagefrequency because lower adhesion to mucus may allow the drug to be moreevenly spread across the surface of the eye, thereby avoiding the eye'snatural clearance mechanisms and prolonging their residence at theocular surface. Improved mucus penetration allows the drug to penetratethrough the mucus coating of the eye more quickly. With respect to theback of the eye, MPPs may allow improved delivery so that atherapeutically effective amount of a drug can reach the back of theeye. In some embodiments, MPPs may effectively penetrate throughphysiological mucus to facilitate sustained drug release directly to theunderlying tissues, as described in more detail below. Mucus-penetratingparticles are further disclosed in US Patent application publications2013/0316009, 2013/01316006, and 2015/0125539, and U.S. Pat. No.9,056,057, incorporated by reference herein for all they discloseregarding mucus-penetrating particles.

Coated Particles

In some embodiments, the particles described herein have a core-shelltype arrangement. The core may comprise any suitable material such as asolid pharmaceutical agent having a relatively low aqueous solubility, apolymeric carrier, a lipid, and/or a protein. The core may also comprisea gel or a liquid in some embodiments. The core may be coated with acoating or shell comprising a mucus penetration-enhancingsurface-altering agent that facilitates mobility of the particle inmucus. As described in more detail below, in some embodiments the mucuspenetration-enhancing surface-altering agent may comprise a polymer(e.g., a synthetic or a natural polymer) having pendant hydroxyl groupson the backbone of the polymer. The molecular weight and/or degree ofhydrolysis of the polymer may be chosen to impart certain transportcharacteristics to the particles, such as increased transport throughmucus. In certain embodiments, the mucus penetration-enhancingsurface-altering agent may comprise a triblock copolymer comprising ahydrophilic block-hydrophobic block-hydrophilic block configuration. Themolecular weights of each of the blocks may be chosen to impart certaintransport characteristics to the particles, such as increased transportthrough mucus. In certain embodiments, the mucus penetration-enhancingsurface-altering agent may comprise a polysorbate.

Some embodiments of a coated particle are depicted in FIG. 1. In FIG. 1,particle 10 includes a core 16 (which may be in the form of a particle)and a coating 20 surrounding the core. The core includes a surface 24 towhich one or more surface-altering agents can be attached or adhered.For instance, in some cases, core 16 is surrounded by coating 20, whichincludes an inner surface 28 and an outer surface 32. The coating maycomprise one or more surface-altering agents 34, such as a polymer(e.g., a block copolymer and/or a polymer having pendant hydroxylgroups), which may associate with surface 24 of the core. Particle 10may optionally include one or more components 40 such as targetingmoieties, proteins, nucleic acids, and bioactive agents which mayoptionally impart specificity to the particle. For example, a targetingagent or molecule (e.g., a protein, nucleic acid, nucleic acid analog,carbohydrate, or small molecule), if present, may aid in directing theparticle to a specific location in the subject's body. The location maybe, for example, a tissue, a particular cell type, or a subcellularcompartment. One or more components 40, if present, may be associatedwith the core, the coating, or both; e.g., they may be associated withsurface 24 of the core, inner surface 28 of the coating, outer surface32 of the coating, and/or embedded in the coating. The one or morecomponents 40 may be associated through covalent bonds, absorption, orattached through ionic interactions, hydrophobic and/or hydrophilicinteractions, electrostatic interactions, van der Waals interactions, orcombinations thereof. In some embodiments, a component may be attached(e.g., covalently) to one or more of the surface-altering agents of thecoated particle.

In certain embodiments, a particle described herein has certain arelative velocity, <V_(mean)>_(rel), which is defined as follows:

$\begin{matrix}{{\text{<}V_{mean}\text{>}_{rel}} = \frac{{\text{<}V_{mean}\text{>}_{Sample}} - {\text{<}V_{mean}\text{>}_{{Negative}\mspace{14mu} {control}}}}{{\text{<}V_{mean}\text{>}_{{Positive}\mspace{14mu} {control}}} - {\text{<}V_{mean}\text{>}_{{Negative}\mspace{14mu} {control}}}}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

where <V_(mean)> is the ensemble average trajectory-mean velocity,V_(mean) is the velocity of an individual particle averaged over itstrajectory, the sample is the particle of interest, the negative controlis a 200 nm carboxylated polystyrene particle, and the positive controlis a 200 nm polystyrene particle densely PEGylated with 2 kDa-5 kDa PEG.

The relative velocity can be measured by a multiple particle trackingtechnique. For instance, a fluorescent microscope equipped with a CCDcamera can be used to capture 15 sec movies at a temporal resolution of66.7 msec (15 frames/sec) under 100× magnification from several areaswithin each sample for each type of particles: sample, negative control,and positive control. The sample, negative and positive controls may befluorescent particles to observe tracking. Alternatively non-fluorescentparticles may be coated with a fluorescent molecule, a fluorescentlytagged surface agent or a fluorescently tagged polymer. An advancedimage processing software (e.g., Image Pro or MetaMorph) can be used tomeasure individual trajectories of multiple particles over a time-scaleof at least 3.335 sec (50 frames).

In some embodiments, a MPP described herein has a relative velocity, ora mean relative velocity, in mucus, of at least about 0.3, about 0.4,about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7,about 1.8, about 1.9, about 2.0; up to: about 10.0, about 8.0, about6.0, about 4.0, about 3.0, about 2.0, about 1.9, about 1.8, about 1.7,about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about 1.1, about1.0, about 0.9, about 0.8, or about 1.7; about 0.5-6, or any velocity ina range bounded by any of these values.

In certain embodiments, an MPP described herein can diffuse throughmucus or a mucosal barrier at a greater rate or diffusivity, or may havea greater geometric mean squared displacement, than a control particleor a corresponding particle (e.g., a corresponding particle that isunmodified and/or is not coated with a coating described herein). Insome cases, a particle described herein may pass through mucus or amucosal barrier at a rate of diffusivity, or with a geometric meansquared displacement, that is at least about 10 times, 20 times, 30times, 50 times, 100 times, 200 times, 500 times, 1000 times, 2000times, 5000 times, 10000 times, or more; up to about 10000 times, about5000 times, about 2000 times, about 1000 times, about 500 times, about200 times, about 100 times, about 50 times, about 30 times, about 20times, about 10 times; about 10-1000 times higher than a controlparticle or a corresponding particle; or may have any increase indiffusivity in a range bounded by any of these values.

In some embodiments, an MPP described herein diffuses through a mucosalbarrier at a rate approaching the rate or diffusivity at which theparticles can diffuse through water. In some cases, a particle describedherein may pass through a mucosal barrier at a rate or diffusivity thatis at least about 1/10,000, about 1/5000, about 1/2000, about 1/1000,about 1/900, about 1/800, about 1/700, about 1/600, about 1/500, about1/400, about 1/300, about 1/200, or about 1/100; up to about 1/100,about 1/200, about 1/300, about 1/400, about 1/500, about 1/600, about1/700, about 1/800, about 1/900, about 1/1000, about 1/2000, about1/5000, about 1/10; or 1/5000-1/500, the diffusivity that the particlediffuses through water under identical conditions, or any rate ordiffusivity in a range bounded by any of these values.

In a particular embodiment, an MPP described herein may diffuse throughhuman mucus at a diffusivity that is less than about 1/500 thediffusivity that the particle diffuses through water. In some cases, themeasurement is based on a time scale of about 1 second, or about 0.5second, or about 2 seconds, or about 5 seconds, or about 10 seconds.

In certain embodiments provided herein particles travel through mucus atcertain absolute diffusivities. For example, the MPPs described hereinmay travel at diffusivities of at least about 1×10⁻⁴ μm/s, 2×10⁻⁴ μm/s,5×10⁻⁴ μm/s, 1×10⁻³ μm/s, 2×10⁻³ μm/s, 5×10⁻³ μm/s, 1×10⁻² μm/s, 2×10⁻²μm/s, 4×10⁻² μm/s, 5×10⁻² μm/s, 6×10⁻² μm/s, 8×10⁻² μm/s, 1×10⁻¹ μm/s,2×10⁻¹ μm/s, 5×10⁻¹ μm/s, 1 μm/s, or 2 μm/s; up to about 2 μm/s, about 1μm/s, about 5×10⁻¹ μm/s, about 2×10⁻¹ μm/s, about 1×10⁻¹ μm/s, about8×10⁻² μm/s, about 6×10⁻² μm/s, about 5×10⁻² μm/s, about 4×10⁻² μm/s,about 2×10⁻² μm/s, about 1×10⁻² μm/s, about 5×10⁻³ μm/s, about 2×10⁻³μm/s, about 1×10⁻³ μm/s, about 5×10⁻⁴ μm/s, about 2×10⁻⁴ μm/s, or about1×10⁻⁴ μm/s; or about 2×10⁻⁴-1×10⁻¹ μm/s, or any absolute diffusivity ina range bounded by any of these values. In some cases, the measurementis based on a time scale of about 1 second, or about 0.5 second, orabout 2 seconds, or about 5 seconds, or about 10 seconds.

In some embodiments, a subject composition comprises a plurality ofparticles coated with a mucus penetration-enhancing coating comprising asurface-altering agent, such as a plurality of coated particles. Such acoated particle contains a core comprising the drug and a coatingcomprising a surface-altering agent.

The surface-altered particles, such as the coated particles describedherein, may have any suitable shape and/or size. In some embodiments, acoated particle has a shape substantially similar to the shape of thecore. In some cases, a coated particle described herein may be ananoparticle, i.e., the particle has a characteristic dimension of lessthan about 1 micrometer, where the characteristic dimension of theparticle is the diameter of a perfect sphere having the same volume asthe particle. In other embodiments, larger sizes are possible. Aplurality of particles, in some embodiments, may also be characterizedby an average size, an average characteristic dimension, an averagelargest cross-sectional dimension, or an average smallestcross-sectional dimension of less than or equal to about 10 μm, lessthan or equal to about 5 μm, less than or equal to about 1 μm, about700-800 nm, about 500-700 nm, about 400-500 nm, about 300-400 nm, about200-300 nm, about 50-200 nm, about 5-100 nm, about 50-75 nm, about 5-50nm, about 5-40 nm, about 5-35 nm, about 5-30 nm, about 5-25 nm, about5-20 nm, about 5-15 nm, about 0.1-5 nm, about 200-400 nm, about 200-500nm, about 100-400 nm, or about 100-300 nm; at least about 5 nm, at leastabout 20 nm, at least about 50 nm, about 100-700 nm, about 200-500 nm,about 5 μm, about 10 nm, about 1 μm, about 10 nm-5 μm, 50-500 nm,200-500 nm, about 1-10 μm or any size in a range bounded by any of thesevalues. In some embodiments, the sizes of the cores formed by a processdescribed herein have a Gaussian-type distribution.

It is appreciated in the art that the ionic strength of a formulationcomprising particles may affect the polydispersity of the particles.Polydispersity is a measure of the heterogeneity of sizes of particlesin a formulation. Heterogeneity of particle sizes may be due todifferences in individual particle sizes and/or to the presence ofaggregation in the formulation. A formulation comprising particles isconsidered substantially homogeneous or “monodisperse” if the particleshave essentially the same size, shape, and/or mass. A formulationcomprising particles of various sizes, shapes, and/or masses is deemedheterogeneous or “polydisperse”.

In some embodiments, the polydispersity index of a subject composition,such as a polydispersity index of a particle size or a molecular weight,is at least about 0.005, about 0.01, about 0.05, about 0.1, about 0.15,about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about0.8, about 0.9, or about 1; up to about 1, about 0.9, about 0.8, about0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.15,about 0.1, about 0.05, about 0.01, or about 0.005; about 0.1-0.5, about0.1, about 0.15, about 0.2, or any polydispersity index in a rangebounded by any of these values. Polydispersity index may be determinedaccording to ISO standards ISO 13321:1996 E and ISO 22412:2008.

Although many methods for determining sizes of particles are known, thesizes described herein (e.g., average particle sizes, thicknesses) referto ones measured by dynamic light scattering.

The MPPs may result in a subject composition that is capable ofsustaining a therapeutically effective level, or delivering atherapeutically effect amount, of the pharmaceutical agent, such as ahydrocortisone derivative, in a target tissue. For example, anophthalmically effective level or an ophthalmically effective amount ofthe drug-containing MPP may be delivered to an ocular tissue, e.g. ananterior ocular tissue, such as a palpebral conjunctiva, a bulbarconjunctiva, a fornix conjunctiva, an aqueous humor, an anterior sclera,a cornea, an iris, or a ciliary body; or the back of the eye, such as avitreous humor, a vitreous chamber, such as a retina, a macula, achoroid, a posterior sclera, a uvea, an optic nerve, or the bloodvessels or nerves which vascularize or innervate a posterior ocularregion or site. In some embodiments, the concentration of thepharmaceutical agent, such as a hydrocortisone derivative, in the tissuemay be increased by at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60% or more, within a short relatively amount of time,compared to the concentration of the pharmaceutical agent whenadministered without the mucus penetration-enhancing coating.

A subject composition may increase the drug level, e.g. thehydrocortisone derivative level, within a relatively short amount oftime, such as within about 24 hours, about 18 hours, about 12 hours,about 9 hours, about 6 hours, about 4 hours, about 3 hours, about 2hours, about 1 hour, about 30 minutes, about 20 minutes, about 10minutes, about 10 minutes to about 2 hours, or any time in a rangebounded by any of these values.

A subject composition may achieve therapeutically effective level or anophthalmically effective level of hydrocortisone derivatives,potentially as a result of the mucus penetration-enhancing coating ofthe MPP, for a sustained period of time after administration, such asleast: 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6hours, 9 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5days, 6 days, or 1 week; up to: 1 week, 6 days, 5 days, 4 days, 3 days,2 days, 1 day, 12 hours, 9 hours, 6 hours, 4 hours, 2 hours, 1 hour; orabout 4 hours to about 1 week, about 10 minutes to about 2 hours, or anytime in a range bounded by any of these values.

The core may contain particles of pharmaceutical agents that have a lowaqueous solubility, such as a hydrocortisone derivative disclosed belowand in U.S. Pat. No. 8,906,892 which is incorporated herein by referencefor all it discloses regarding hydrocortisone derivatives. Thehydrocortisone derivative may be in a crystalline or nanocrystalline(including any polymorph form) or an amorphous form. In someembodiments, the hydrocortisone derivative is(10R,11S,13S,17R)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl3-(phenylsulfonyl)propanoate (Compound 1),(10R,11S,13S,17R)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-ylfuran-2-carboxylate (Compound 2), or(10R,11S,13S,17R)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl2-(4-bromophenyl)acetate (Compound 3).

Unless otherwise indicated, any reference to a compound herein, such asa hydrocortisone derivative, by structure, name, or any other means,includes prodrugs, such as ester prodrugs; alternate solid forms, suchas polymorphs, solvates, hydrates, etc.; tautomers; or any otherchemical species that may rapidly convert to a compound described hereinunder conditions in which the compounds are used as described herein.

The core may comprise the pharmaceutical agent, such as a hydrocortisonederivative. The core may be substantially all pharmaceutical agent, ormay comprise additional components, such as a polymer, a lipid, aprotein, a gel, a liquid, a surfactant, a tonicity agent (such asglycerin), a buffer, a salt (such as NaCl), a preservative (such asbenzalkonium chloride), a chelating agent (such as EDTA), a filler, etc.In some embodiments, the core particles comprise a hydrocortisonederivative that is encapsulated in a polymer, a lipid, a protein, or acombination thereof. In various embodiments the term encapsulationencompasses any or all of a coating or shell of the encapsulatingsubstance surrounding the rest of the core particle, a solidifiedco-solution comprising the encapsulating substance and thehydrocortisone derivative of the core particle, a dispersion of thehydrocortisone derivative within a matrix comprising the encapsulatingsubstance, and the like.

In embodiments in which the core particles comprise relatively highamounts of a hydrocortisone derivative disclosed herein (e.g., at leastabout 50 wt % of the core particle), the core particles generally havean increased loading of a hydrocortisone derivative compared toparticles that are formed by encapsulating agents into polymericcarriers. This is an advantage for drug delivery applications, sincehigher drug loadings mean that fewer numbers of particles may be neededto achieve a desired effect compared to the use of particles containingpolymeric carriers.

Suitable polymers for use in a core may include a synthetic polymer,e.g. non-degradable polymers such as polymethacrylate and degradablepolymers such as polylactic acid, polyethylene glycol, polyglycolic acidand copolymers thereof (such as PLA-PEG), and/or a natural polymer, suchas hyaluronic acid, chitosan, and collagen, or a mixture of polymers.

A core may comprise a biodegradable polymer such as poly(ethyleneglycol)-poly(propylene oxide)-poly(ethylene glycol) triblock copolymers,poly(lactide) (or poly(lactic acid)), poly(glycolide) (or poly(glycolicacid)), poly(orthoesters), poly(caprolactones), polylysine,poly(ethylene imine), poly(acrylic acid), poly(urethanes),poly(anhydrides), poly(esters), poly(trimethylene carbonate),poly(ethyleneimine), poly(acrylic acid), poly(urethane), poly(beta aminoesters) or the like, and combinations, copolymers or derivatives ofthese and/or other polymers, for example, poly(lactide-co-glycolide)(PLGA).

In certain embodiments, a polymer may biodegrade within a period that isacceptable in the desired application. In certain embodiments, such asin vivo therapy, such degradation occurs in a period usually less thanabout five years, one year, six months, three months, one month, fifteendays, five days, three days, or even one day or less (e.g., 1-4 hours,4-8 hours, 4-24 hours, 1-24 hours) on exposure to a physiologicalsolution with a pH between 6 and 8 having a temperature of between 25and 37° C. In some embodiments, the polymer degrades in a period ofbetween about one hour and several weeks.

The pharmaceutical agent may be present in the core in any suitableamount, e.g., at about 1-100 wt %, 5-100 wt %, 10-100 wt %, 20-100 wt %,30-100 wt %, 40-100 wt %, 50-100 wt %, 60-100 wt %, 70-100 wt %, 80-100wt %, 85-100 wt %, 90-100 wt %, 95-100 wt %, 99-100 wt %, 50-90 wt %,60-90 wt %, 70-90 wt %, 80-90 wt %, 85-90 wt % of the core, 70 wt %, 75wt %, 80 wt %, 85 wt %, 90 wt %, 95 wt %, 97 wt %, or any amount in arange bounded by any of these values.

If a polymer is present in the core, the polymer may be present in thecore in any suitable amount, e.g., 1-20%, 20-40%, 40-60%, 60-80%, or80-95% by weight, or any amount in a range bounded by any of thosevalues. In one set of embodiments, the core is formed is substantiallyfree of a polymeric component.

The core may have any suitable shape and/or size. For instance, the coremay be substantially spherical, non-spherical, oval, rod-shaped,pyramidal, cube-like, disk-shaped, wire-like, or irregularly shaped. Thecore may have a largest or smallest cross-sectional dimension of, forexample, less than or equal to: about 10 μm, about 5 μm, about 1 μm,about 5-800 nm, about 5-700 nm, about 5-500 nm, about 400 nm, or about300 nm; 5-200 nm, 5-100 nm, 5-75 nm, 5-50 nm, 5-40 nm, 5-35 nm, 5-30 nm,5-25 nm, 5-20 nm, 5-15 nm, about 50-500 nm, at least: about 20 nm, about50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, at leastabout 500 nm, about 1 μm, or about 5 μm, or any size in a range boundedby any of these values. In some embodiments, the sizes of the coresformed by a process described herein have a Gaussian-type distribution.

The surface of a core may be partially or completely covered by a mucuspenetration-enhancing coating. The coating may comprise asurface-altering agent, which may be any agent that modifies the surfaceof the core particles to reduce the adhesion of the particles to mucusand/or to facilitate penetration of the particles through physiologicalmucus.

In some embodiments, hydrophobic portions of a mucuspenetration-enhancing surface-altering agent (e.g., non-hydrolyzedportions of polyvinyl alcohol, hydrophobic polyalkylene oxide, etc.) mayallow the polymer to be adhered to the core surface (e.g., in the caseof the core surface being hydrophobic), thus allowing for a strongassociation between the core and the polymer.

In some embodiments, hydrophilic portions of a surface-altering agent(e.g. hydrolyzed portions of polyvinyl alcohol, polethylene oxide, etc.)can render the surface-altering agent, and as a result the particle,hydrophilic. The hydrophilicity may shield the coated particles fromadhesive interactions with mucus, which may help to improve mucustransport or penetration.

Examples of suitable surface-altering agents include a block copolymerhaving one or more relatively hydrophilic blocks and one or morerelatively hydrophobic blocks, such as a triblock copolymer, wherein thetriblock copolymer comprises a hydrophilic block-hydrophobicblock-hydrophilic block configuration, a diblock copolymer having ahydrophilic block-hydrophobic block configuration; a combination of ablock copolymer with one or more other polymers suitable for use in acoating; a polymer-like molecule having a nonlinear blockconfigurations, such as nonlinear configurations of combinations ofhydrophilic and hydrophobic blocs, such as a comb, a brush, or a starcopolymer; a synthetic polymer having pendant hydroxyl groups on thebackbone of the polymer; a polysorbate; a surfactant; etc.

The surface-altering agent may have any suitable molecular weight, suchas at least about 1 kDa, about 2 kDa, about 4 kDa, about 5 kDa, about 8kDa, about 9 kDa, about 10 kDa, about 12 kDa, about 15 kDa about 20 kDa,about 25 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa,about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa about 110 kDa,about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 200kDa, about 500 kDa, or about 1000 kDa; less than or equal to about 1000kDa, about 500 kDa, about 200 kDa, about 180 kDa, about 150 kDa, about130 kDa, about 120 kDa, about 100 kDa, about 85 kDa, about 70 kDa, about65 kDa, about 60 kDa, about 50 kDa, about 40 kDa, about 30 kDa, about 20kDa, about 15 kDa, about 10 kDa; about 10-30 kDa, about 1-100 kDa, about1-50 kDa, about 1-3 kDa, about 2-7 kDa, about 5-10 kDa, about 8-12 kDa,about 9-15 kDa, about 10-15 kDa, about 12-17 kDa, about 15-25 kDa about20-30 kDa, about 25-40 kDa, about 30-50 kDa, about 40-60 kDa, about50-70 kDa; or a molecular weight in a range bounded by any of thesevalues.

When the surface-altering agent is a block copolymer, the molecularweight of the hydrophilic blocks and the hydrophobic blocks of the blockcopolymers, or the relative amount of the hydrophobic block with respectto the hydrophilic block, may affect the mucoadhesion and/or mucuspenetration of a core and association of the block copolymer with thecore. Many block copolymers comprise a polyether portion, such as apolyalkylether portion. A polyether block may be relatively hydrophilic(e.g. polyethylene glycol) or relatively hydrophobic (e.g. polyalkyleneglycols based upon monomer or repeating units having 3 or more carbonatoms).

The copolymer may have any suitable molecular weight, such as at leastabout 1 kDa, about 2 kDa, about 4 kDa, about 5 kDa, about 8 kDa, about 9kDa, about 10 kDa, about 12 kDa, about 15 kDa about 20 kDa, about 25kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70kDa, about 80 kDa, about 90 kDa, about 100 kDa about 110 kDa, about 120kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 200 kDa, about500 kDa, or about 1000 kDa; less than or equal to about 1000 kDa, about500 kDa, about 200 kDa, about 180 kDa, about 150 kDa, about 130 kDa,about 120 kDa, about 100 kDa, about 85 kDa, about 70 kDa, about 65 kDa,about 60 kDa, about 50 kDa, about 40 kDa, about 30 kDa, about 20 kDa,about 15 kDa, about 10 kDa; about 10-30 kDa, about 1-100 kDa, about 1-50kDa, about 1-3 kDa, about 2-7 kDa, about 5-10 kDa, about 8-12 kDa, about9-15 kDa, about 10-15 kDa, about 12-17 kDa, about 15-25 kDa about 20-30kDa, about 25-40 kDa, about 30-50 kDa, about 40-60 kDa, about 50-70 kDa;or a molecular weight in a range bounded by any of these values.

A hydrophobic block may be any suitable block in a block copolymer thatis relatively hydrophobic as compared to another block in the copolymer.The hydrophobic block may be substantially present in the interior ofthe coating and/or at the surface of the core particle, e.g., tofacilitate attachment of the coating to the core. Examples of suitablepolymers for use in the hydrophobic block include polyalkylethers having3 or more carbon atoms in each repeating unit, such as polypropyleneglycol, polybutylene glycol, polypentylene glycol, polyhexylene glycol,etc.; esters of polyvinyl alcohol such as polyvinyl acetate; polyvinylalcohol having a low degree of hydrolysis, etc.

Any suitable amount of the hydrophobic blocks may be used. For example,the hydrophobic block may be a sufficiently large portion of the polymerto allow the polymer to adhere to the core surface, particularly if thecore surface is hydrophobic. In certain embodiments, the molecularweight of the (one or more) relatively hydrophobic blocks of a blockcopolymer, such as poly(propylene oxide) (PPO), is at least about 0.5kDa, about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa,about 6 kDa, about 10 kDa, about 12 kDa, about 15 kDa, about 20 kDa,about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa,about 100 kDa about 110 kDa, about 120 kDa, about 130 kDa, about 140kDa, about 150 kDa, about 200 kDa, about 500 kDa, about 1000 kDa; up toabout 1000 kDa, about 500 kDa, about 200 kDa, about 150 kDa, about 140kDa, about 130 kDa, about 120 kDa, about 110 kDa, about 100 kDa, about90 kDa, about 80 kDa, about 50 kDa, about 20 kDa, about 15 kDa, about 13kDa, about 12 kDa, about 10 kDa, about 8 kDa, or about 6 kDa; or about3-15 kDa, 0.5-5 kDa, 0.5-1 kDa, 1-2 kDa, 2-3 kDa, 2-2.5 kDa, 2.5-3 kDa,3-8 kDa, 3-3.5 kDa, 3.5-4 kDa, 3-4 kDa, 4-5 kDa, about 0.5-3 kDa, 2.5-3kDa, 2.7-3 kDa, 2.8-3 kDa, 3-3.3 kDa, 3-3.5 kDa, 3.5-3.7 kDa, 3.5-4 kDa,5-4.5 kDa, 5-10 kDa, or any molecular weight in a range bounded by anyof these values.

A hydrophilic block may be any suitable block in a block copolymer thatis relatively hydrophilic as compared to another block in the blockcopolymer. In some cases, the hydrophilic blocks may be substantiallypresent at the outer surface of the particle. For example, thehydrophilic blocks may form a majority of the outer surface of thecoating and may help stabilize the particle in an aqueous solutioncontaining the particle. Examples of suitable polymers for use in thehydrophilic block include polyethylene glycol, or synthetic polymershaving hydroxyl pendant groups such as polyvinyl alcohol having a highdegree of hydrolysis. Any suitable amount of the hydrophilic block maybe used, such as an amount that is sufficiently large to render thecoated particle hydrophilic when present at the surface of the particle.

In some embodiments, the combined (one or more) relatively hydrophilicblocks, e.g. PEO or polyvinyl alcohol, or repeat units of a blockcopolymer constitute at least about 10 wt %, about 15 wt %, about 20 wt%, about 25 wt %, about 30 wt %, about 35 wt %, about 40 wt %, about 45wt %, about 50 wt %, about 55 wt %, about 60 wt %, about 65 wt %, orabout 70 wt %; up to about 90 wt %, about 80 wt %, about 60 wt %, about50 wt %, or about 40 wt % of the block copolymer; or about 30-80 wt %,about 10-30 wt %, 10-40 wt %, about 30-50 wt %, about 40-80 wt %, about50-70 wt %, about 70-90 wt %, about 15-80 wt %, about 20-80 wt %, about25-80 wt %, about 30-80 wt %, of the block copolymer, or any percentagein a range bounded by any of these values.

In some embodiments, the molecular weight of the (one or more)relatively hydrophilic blocks or repeat units, such as poly(ethyleneoxide) (PEO) or poly(vinyl alcohol) (PVA), of the block copolymer may beat least about 0.5 kDa, about 1 kDa, about 2 kDa, about 3 kDa, about 4kDa, about 5 kDa, about 6 kDa, about 10 kDa, about 12 kDa, about 15 kDa,about 20 kDa, or about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa,about 90 kDa, about 100 kDa about 110 kDa, about 120 kDa, about 130 kDa,about 140 kDa, about 150 kDa, about 200 kDa, about 500 kDa, or about1000 kDa; up to about 1000 kDa, about 500 kDa, about 200 kDa, about 150kDa, about 140 kDa, about 130 kDa, about 120 kDa, about 110 kDa, about100 kDa, about 90 kDa, about 80 kDa, about 50 kDa, about 20 kDa, about15 kDa, about 13 kDa, about 12 kDa, about 10 kDa, about 8 kDa, about 6kDa, about 5 kDa, about 3 kDa, about 2 kDa, about 1 kDa; about 1-2 kDa,about 2-4 kDa, about 3-15 kDa, about 4-7 kDa, 7-10 kDa, about 10-12 kDa,about 10-15 kDa, or any molecular weight in a range bounded by any ofthese values.

In embodiments in which two hydrophilic blocks flank a hydrophobicblock, the molecular weights, and the chemical identity, of the twohydrophilic blocks may be substantially the same or different.

In certain embodiments, the polymer is a triblock copolymer of apolyalkyl ether (e.g., polyethylene glycol, polypropylene glycol) andanother polymer (e.g., a synthetic polymer having pendant hydroxylgroups on the backbone of the polymer (e.g., PVA). In certainembodiments, the polymer is a triblock copolymer of a polyalkyl ether(such as polyethylene glycol) and another polyalkyl ether. In certainembodiments, the polymer includes a polypropylene glycol unit flanked bytwo more hydrophilic units. In certain embodiments, the polymer includestwo polyethylene glycol units flanking a more hydrophobic unit. Themolecular weights of the two blocks flanking the central block may besubstantially the same or different.

In certain embodiments, the polymer is of Formula 1:

With respect to Formula 1, m is 2-1730, 5-70, 5-100, 20-100, 10-20,20-30, 30-40, 40-50, 50-60, 60-70, 10-50, 40-60, 50-70, 50-100, 100-300,300-500, 500-700, 700-1000, 1000-1300, 1300-1600, 1600-2000, about 15,about 20, about 31, about 41, about 51, about 61, about 68, or anyinteger in a range bounded by any of these values.

With respect to Formula 1, n¹ and n² may be the same or different. Insome embodiments, n¹+n², is 2-1140, 2-10, 10-30, 30-40, 40-70, 70-150,150-200, 10-170, 50-150, 90-110, 100-200, 200-400, 400-600, 600-800,800-1000, 1000-1500, about 2, about 6, about 8, about 9, about 18, about29, about 35, about 39, about 41, about 68, about 82, about 127, about164, about 191, or any integer in a range bounded by any of thesevalues. In certain embodiments, n¹+n² is at least 2 times m, 3 times m,or 4 times m.

With respect to Formula 1, in some embodiments m is about 10-30 andn¹+n² is about 2-10, m is about 10-30 and n¹+n² is about 10-30, m isabout 30-50 and n¹+n² is about 2-10, m is about 40-60 and n¹+n² is about2-10, m is about 30-50 and n¹+n² is about 40-100, m is about 60-80 andn¹+n² is about 2-10, m is about 40-60 and n¹+n² is about 20-40, m isabout 10-30 and n¹+n² is about 10-30, m is about 60-80 and n¹+n² isabout 20-40, m is about 40-60 and n¹+n² is about 40-100, m is about30-50 and n¹+n² is about 100-200, m is about 30-50 and n¹+n² is about100-200, m is about 60-80 and n¹+n² is about 100-200, or m is about60-80 and n¹+n² is about 20-40.

In certain embodiments, the coating includes a surface-altering agentcomprising a (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (hereinafter“PEG-PPO-PEG triblock copolymer”), present in the coating alone or incombination with another polymer such as a synthetic polymer havingpendant hydroxyl groups on the backbone of the polymer (e.g., PVA). Asdescribed herein, the PEG blocks may be interchanged with PEO blocks insome embodiments. The molecular weights of the PEG (or PEO) and PPOsegments of the PEG-PPO-PEG triblock copolymer may be selected so as toreduce the mucoadhesion of the particle, as described herein. Withoutwishing to be bound by theory, a particle having a coating comprising aPEG-PPO-PEG triblock copolymer may have reduced mucoadhesion as comparedto a control particle due to, at least in part, the display of aplurality of PEG (or PEO) segments on the particle surface. The PPOsegment may be adhered to the core surface (e.g., in the case of thecore surface being hydrophobic), thus allowing for a strong associationbetween the core and the triblock copolymer. In some cases, thePEG-PPO-PEG triblock copolymer is associated with the core throughnon-covalent interactions. For purposes of comparison, the controlparticle may be, for example, a carboxylate-modified polystyreneparticle of similar size as the coated particle in question.

In some embodiments, a triblock copolymer, such as a PEO-PPO-PEOcopolymer, has an average molecular weight that is at least about 1 kDa,about 2 kDa, about 4 kDa, about 5 kDa, about 8 kDa, about 9 kDa, about10 kDa; less than or equal to about 100 kDa, about 50 kDa, about 20 kDa,about 15 kDa, about 10 kDa; or is about 1-3 kDa, 1-3 kDa, 2-4 kDa, 3-5kDa, 4-6 kDa, 5-7 kDa, 6-8 kDa, 7-9 kDa, 8-10 kDa, 5-7 kDa, about 2-7kDa, about 5-10 kDa, about 8-12 kDa, about 9-15 kDa, about 10-15 kDa,about 12-17 kDa, about 15-25 kDa about 20-30 kDa, about 25-40 kDa, about30-50 kDa, about 40-60 kDa, about 50-70 kDa; or a molecular weight in arange bounded by any of these values.

In certain embodiments, a surface-altering agent includes a polymercomprising a poloxamer, having the trade name Pluronic®. Pluronic®polymers that may be useful in the embodiments described herein include,but are not limited to, F127, F38, F108, F68, F77, F87, F88, F98, F123,L101, L121, L31, L35, L43, L44, L61, L62, L64, L81, L92, N3, P103, P104,P105, P123, P65, P84, and P85.

In some embodiments, the surface-altering agent comprises Pluronic F127,F108, P123, P105, or P103.

Examples of molecular weights of certain Pluronic® molecules are shownin Table 1.

TABLE 1 Molecular Weights of Pluronic ® molecules Pluronic ® PoloxamerAverage MW MW PPO PEO wt % MW PEO L31 101 1000 900 10 100 L44 124 20001200 40 800 L81 231 2667 2400 10 267 L101 331 3333 3000 10 333 P65 1853600 1800 50 1800 L121 401 4000 3600 10 400 P103 333 4286 3000 30 1286F38 108 4500 900 80 3600 P105 335 6000 3000 50 3000 F87 237 8000 2400 705600 F68 188 9000 1800 80 7200 F127 407 12000 3600 70 8400 P123 403 57504030 30 1730 F108 338 14600 3250 80 11350

A surface-altering agent may include a synthetic polymer having pendanthydroxyl groups on the backbone of the polymer, such as a poly(vinylalcohol), a partially hydrolyzed poly(vinyl acetate), a copolymer ofvinyl alcohol and vinyl acetate, a poly(ethylene glycol)-poly(vinylacetate)-poly(vinyl alcohol) copolymer, a poly(ethyleneglycol)-poly(vinyl alcohol) copolymer, a poly(propyleneoxide)-poly(vinyl alcohol) copolymer, a poly(vinyl alcohol)-poly(acrylamide) copolymer, etc.

The synthetic polymer described herein (e.g., one having pendanthydroxyl groups on the backbone of the polymer) may have any suitablemolecular weight, such as at least about 1 kDa, about 2 kDa, about 5kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 12 kDa, about 15 kDaabout 20 kDa, about 25 kDa, about 30 kDa, about 40 kDa, about 50 kDa,about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa,about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150kDa, about 200 kDa, about 500 kDa, or about 1000 kDa; up to about 1000kDa, about 500 kDa, about 200 kDa, about 180 kDa, about 150 kDa, about130 kDa, about 120 kDa, about 100 kDa, about 85 kDa, about 70 kDa, about65 kDa, about 60 kDa, about 50 kDa, about 40 kDa, about 30 kDa, about 20kDa, about 15 kDa, or about 10 kDa; about 1-1000 kDa, about 1-10 kDa,about 5-20 kDa, about 10-30 kDa, about 20-40 kDa, about 30-50 kDa, about40-60 kDa, about 50-70 kDa, about 60-80 kDa, about 70-90 kDa, about80-100 kDa, about 90-110 kDa, about 100-120 kDa, about 110-130 kDa,about 120-140 kDa, about 130-150 kDa, about 140-160 kDa, about 150-170kDa, or any molecular weight in a range bounded by any of these values.

Poly(vinyl alcohol) may be prepared by polymerizing a vinyl ester toproduce a poly(vinyl ester), such as poly(vinyl acetate), and thenhydrolyzing the ester to leave free pendant hydroxy groups. Partiallyhydrolyzed PVA comprises two types of repeating units: vinyl alcoholunits (which are relatively hydrophilic) and residual vinyl acetateunits (which are relatively hydrophobic). Some embodiments may includeone or more blocks of vinyl alcohol units and one or more blocks ofvinyl acetate units. In certain embodiments, the repeat units form acopolymer, e.g., a diblock, triblock, alternating, or random copolymer.

The amount of hydrolysis, or the percentage of vinyl alcohol units ascompared to the total number of vinyl alcohol+vinyl acetate units, mayaffect or determine the relative hydrophilicity or hydrophobicity of apoly(vinyl alcohol), and can affect the mucus penetration of theparticles. It may be helpful for the degree of hydrolysis to be lowenough to allow sufficient adhesion between the PVA and the core (e.g.,in the case of the core being hydrophobic). It may also be helpful forthe degree of hydrolysis to be high enough to enhance particle transportin mucus. The appropriate level of hydrolysis may depend on additionalfactors such as the molecular weight of the polymer, the composition ofthe core, the hydrophobicity of the core, etc.

Less than 95% hydrolysis in a poly(vinyl alcohol) may render a particlemucus penetrating. In some embodiments, a synthetic polymer (e.g., PVAor partially hydrolyzed poly(vinyl acetate) or a copolymer of vinylalcohol and vinyl acetate) may be at least: about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 87%, about 90%, about 95%, orabout 98% hydrolyzed; up to about 100%, about 98%, about 97%, about 96%,about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about87%, about 85%, about 80%, about 75%, about 70%, or about 60%hydrolyzed; about 80-95%, about 30-95%, about 70-94%, about 30-95%, orabout 70-94% hydrolyzed, or any percentage in a range bounded by any ofthese values.

In some embodiments, a synthetic polymer described herein is, orcomprises, PVA. PVA is a non-ionic polymer with surface activeproperties. In some embodiments, the hydrophilic units of a syntheticpolymer described herein may be substantially present at the outersurface of the particle.

The molar fraction of the relatively hydrophilic units and therelatively hydrophobic units of a synthetic polymer may be selected soas to reduce the mucoadhesion of a core and to ensure sufficientassociation of the polymer with the core, respectively. The molarfraction of the relatively hydrophilic units to the relativelyhydrophobic units of a synthetic polymer may be, for example, 0.5:1(hydrophilic units:hydrophobic units), 1:1, 2:1, 3:1, 5:1, 7:1, 10:1,15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 75:1, 100:1; up to 100:1, 75:1,50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 7:1, 5:1, 3:1, 2:1, or 1:1;2:1-4:1, 3:1-5:1, 4:1-6:1, 5:1-7:1, 6:1-8-1, 7:1-9:1, 8:1-10:1,9:1-11:1, 10:1-20:1, 15:1-50:1, 20:1-1000:1, or any molar ratio in arange bounded by any of these values.

Examples of PVA polymers having various molecular weights and degree ofhydrolysis are shown in Table 2. The molecular weight (MW) andhydrolysis degree values were provided by the manufacturers.

TABLE 2 Exemplary PVAs. PVA acronym* MW, kDa Hydrolysis degree, % 2K75 275-79 9K80  9-10 80 13K87 13-23 87-89 13K98 13-23 98 31K87 31-50 87-8931K98 31-50 98-99 57K86 57-60 86-89 85K87  85-124 87-89 85K99  85-124 99+ 95K95 95 95 105K80 104 80 130K87 130 87-89 *PVA acronym: XXKYY,where XX stands for the PVA's lower-end molecular weight in kDa and YYstands for the PVA's lower-end hydrolysis in %.

In certain embodiments, the synthetic polymer is represented by Formula2:

With respect to Formula 2 above, m is 0-11630. Similarly, the value of mmay vary. For instance, in certain embodiments, m is at least 5, 10, 20,30, 50, 70, 100, 150, 200, 250, 300, 350, 400, 500, 800, 1000, 1200,1500, 1800, 2000, 2200, 2400, 2600, 3000, 5000, 10000, or 15000; up to15000, 10000, 5000, 3000, 2800, 2400, 2000, 1800, 1500, 1200, 1000, 800,500, 400, 350, 300, 250, 200, 150, 100, 70, 50, 30, 20, or 10; 5-200,10-100, 100-150, 150-200, 200-300, 300-400, 400-600, 600-800, 800-1000,1000-1200, 1200-1400, about 20, about 92, about 102, about 140, about148, about 247, about 262, about 333, about 354, about 538, about 570,about 611, about 643, about 914, about 972, about 1061, about 1064,about 1333, about 1398, about 1418, or any integer in a range bounded byany of these values.

With respect to Formula 2 above, n is 0-22730. In some embodiments, n isat least 5, 10, 20, 30, 50, 100, 200, 300, 500, 800, 1000, 1200, 1500,1800, 2000, 2200, 2400, 2600, 3000, 5000, 10000, 15000, 20000, or 25000;up to 30000, 25000, 20000, 25000, 20000, 15000, 10000, 5000, 3000, 2800,2400, 2000, 1800, 1500, 1200, 1000, 800, 500, 300, 200, 100, or 50;25-20600, 50-2000, 5-1100, 0-400, 1-400; or 1-10, 10-20, 20-30, 30-50,50-80, 80-100, 100-150, 150-200, 200-300, about 3, about 5, about 6,about 9, about 10, about 14, about 19, about 23, about 26, about 34,about 45, about 56, about 73, about 87, about 92, about 125, about 182,about 191, about 265, or any integer in a range bounded by any of thesevalues.

It is noted that n and m may represent the total content of the vinylalcohol and vinyl acetate repeat units in the polymer, or may representblock lengths.

With respect to Formula 2, above, in some embodiments m is about 1-100and n is about 1-10, m is about 1-100 and n is about 20-30, m is about100-200 and n is about 20-30, m is about 100-200 and n is about 10-20, mis about 200-300 and n is about 30-50, m is about 100-200 and n is about1-10, m is about 200-300 and n is about 1-10, m is about 300-500 and nis about 30-50, m is about 500-700 and n is about 70-90, m is about300-500 and n is about 1-10, m is about 500-700 and n is about 1-10, mis about 500-700 and n is about 70-90, m is about 500-700 and n is about90-150, m is about 700-100 and n is about 90-150, m is about 1000-1200and n is about 150-200, m is about 700-100 and n is about 1-10, m isabout 1200-1500 and n is about 10-20, m is about 1000-1200 and n isabout 50-70, m is about 1000-1200 and n is about 200-300, or m is about1200-1500 and n is about 150-200.

In some embodiments, the PVA is PVA2K75, PVA9K80, PVA13K87, PVA31K87,PVA57K86, PVA85K87, PVA105K80, or PVA130K87. The PVA acronyms aredescribed using the formula PVAXXKYY, where XX stands for the PVA'slower-end molecular weight in kDa and YY stands for the PVA's lower-endhydrolysis in %.

A surface-altering agent may include a polysorbate. Examples ofpolysorbates include polyoxyethylene sorbitan monooleate (e.g., Tween®80), polyoxyethylene sorbitan monostearate (e.g., Tween® 60),polyoxyethylene sorbitan monopalmitate (e.g., Tween® 40), andpolyoxyethylene sorbitan monolaurate (e.g., Tween® 20).

In some embodiments, the surface-altering agent comprises a poloxamer, apoly(vinyl alcohol), a polysorbate, or a combination thereof.

In some embodiments, the surface-altering agent comprisesL-α-phosphatidylcholine (PC), 1,2-dipalmitoylphosphatidycholine (DPPC),oleic acid, sorbitan trioleate, sorbitan mono-oleate, sorbitanmonolaurate, a polyoxylene sorbitan fatty acid ester (Tweens), apolysorbate (e.g., polyoxyethylene sorbitan monooleate) (e.g., Tween®80), polyoxyethylene sorbitan monostearate (e.g., Tween® 60),polyoxyethylene sorbitan monopalmitate (e.g., Tween® 40),polyoxyethylene sorbitan monolaurate (e.g., Tween® 20), naturallecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether,lauryl polyoxyethylene ether, polyoxylene alkyl ethers, a blockcopolymer of oxyethylene and oxypropylene, apolyoxyethylene stearate,polyoxyethylene castor oil and/or a derivative thereof, a Vitamin-E-PEGor a derivative thereof, synthetic lecithin, diethylene glycol dioleate,tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glycerylmonooleate, glyceryl monostearate, glyceryl monoricinoleate, cetylalcohol, stearyl alcohol, polyethylene glycol, cetyl pyridiniumchloride, benzalkonium chloride, olive oil, glyceryl monolaurate, cornoil, cotton seed oil, sunflower seed oil, or a derivative and/orcombination thereof.

The surface-altering agent may be present in the pharmaceuticalcomposition in any suitable amount, such as an amount between about0.001-5%, about 0.001-1%, about 1-2%, about 2-3%, about 3-4%, or about4-5% by weight.

The surface-altering agent may be present in any suitable amount withrespect to the pharmaceutical agent. In some embodiments, the ratio ofsurface-altering agent to pharmaceutical agent may be at least about0.001:1 (weight ratio, molar ratio, or w:v ratio), about 0.01:1, about0.01:1, about 1:1, about 2:1, about 3:1, about 5:1, about 10:1, about25:1, about 50:1, about 100:1, or about 500:1. In some embodiments, theratio of surface-altering agent to pharmaceutical agent) is up to about1000:1 (weight ratio, molar ratio, or w:v ratio), about 500:1, about100:1, about 75:1, about 50:1, about 25:1, about 10:1, about 5:1, about3:1, about 2:1, about 1:1, about 0.1:1; and/or about 5:1-50:1, or anyratio in a range bounded by any of these values.

Typically, a coating may be on the surface of, or partially orcompletely surround or coat, the core. In some embodiments, thesurface-altering agent may surround the core particle.

The coating may adhere, or be covalently or non-covalently bound orotherwise attached, to the core. For example, the surface-altering agentmay be covalently attached to a core particle, non-covalently attachedto a core particle, adsorbed to a core, or coupled or attached to thecore through ionic interactions, hydrophobic and/or hydrophilicinteractions, electrostatic interactions, van der Waals interactions, orcombinations thereof. A surface-altering agent may be oriented in aparticular configuration in the coating of the particle. For example, insome embodiments in which a surface-altering agent is a triblockcopolymer, such as a triblock copolymer having a hydrophilicblock-hydrophobic block-hydrophilic block configuration, and thehydrophobic block may be oriented towards the surface of the core, andthe hydrophilic blocks may be oriented away from the core surface (e.g.,towards the exterior of the particle).

The coating may include one layer of material (e.g., a monolayer), ormultilayers of materials. A single type of surface-altering agent may bepresent, or multiple types of surface-altering agent.

The surface-altering agent may be present on the surfaces of the coreparticles at any density that is effective to reduce adhesion to mucusor improved penetration of the particles through mucus. For example, thesurface-altering agent may be present on the surfaces of the coreparticles at a density of at least: about 0.001, about 0.002, about0.005, about 0.01, about 0.02, about 0.05, about 0.1, about 0.2, about0.5, about 1, about 2, about 5, about 10, about 20, about 50, or about100; up to: about 100, about 50, about 20, about 10, about 5, about 2,about 1, about 0.5, about 0.2, about 0.1, about 0.05, about 0.02, orabout 0.01; or about 0.01-1 units or molecules/nm²; or any density in arange bounded by any of these values.

Those of ordinary skill in the art will be aware of methods to estimatethe average density of surface-altering moieties on the core particle(see, for example, S. J. Budijono et al., Colloids and Surfaces A:Physicochem. Eng. Aspects 360 (2010) 105-110 and Joshi, et al., Anal.Chim. Acta 104 (1979) 153-160, each of which is incorporated herein byreference). For example, as described herein, the average density ofsurface-altering moieties can be determined using HPLC quantitation andDLS analysis. A suspension of particles for which surface densitydetermination is of interest is first sized using DLS: a small volume isdiluted to an appropriate concentration (˜100 μg/mL, for example), andthe z-average diameter is taken as a representative measurement ofparticle size. The remaining suspension is then divided into twoaliquots. Using HPLC, the first aliquot is assayed for the totalconcentration of core material and for the total concentration ofsurface-altering moiety. Again using HPLC, the second aliquot is assayedfor the concentration of free or unbound surface-altering moiety. Inorder to get only the free or unbound surface-altering moiety from thesecond aliquot, the particles, and therefore any bound surface-alteringmoiety, are removed by ultracentrifugation. By subtracting theconcentration of the unbound surface-altering moiety from the totalconcentration of surface-altering moiety, the concentration of boundsurface-altering moiety can be determined. Since the total concentrationof core material was also determined from the first aliquot, the massratio between the core material and the surface-altering moiety can bedetermined. Using the molecular weight of the surface-altering moietythe number of surface-altering moiety to mass of core material can becalculated. To turn this number into a surface density measurement, thesurface area per mass of core material needs to be calculated. Thevolume of the particle is approximated as that of a sphere with thediameter obtained from DLS allowing for the calculation of the surfacearea per mass of core material. In this way the number ofsurface-altering moieties per surface area can be determined.

An example of calculating this surface density is presented in Example 5below using the surface area of a perfect sphere with the diameter ofthe core particles determined by dynamic light scattering. Inalternative embodiments surface area is measured as theBrunauer-Emmett-Teller specific surface area which is based on theadsorption of gas molecules to solid surfaces. Most typically nitrogenis the gas used.

In certain embodiments in which the surface-altering agent is adsorbedonto a surface of a core, the surface-altering agent may be inequilibrium with other molecules of the surface-altering agent insolution. In some cases, the adsorbed surface-altering agent may bepresent on the surface of the core at a density described herein.

A coating comprising a surface-altering agent may partially orcompletely surround the core. For example, the coating may surround atleast about 10%, at least about 30%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 99%, up to about 100%, up to about 90%, up to about 80%, upto about 70%, up to about 60%, or up to about 50%, about 80-100% of thesurface area of a core, or any percentage in a range bounded by any ofthese values.

A coating of a particle can have any suitable thickness. For example, acoating may have an average thickness of at least about 1 nm, about 5nm, about 10 nm, about 30 nm, about 50 nm, about 100 nm, about 200 nm,about 500 nm, about 1 μm, or about 5 μm. In other embodiments, thecoating may have an average thickness of up to about 5 μm, about 1 μm,about 500 nm, about 200 nm, about 100 nm, about 50 nm, about 30 nm,about 10 nm, or about 5 nm. In other embodiments, the coating may havean average thickness of about 1-100 nm, or any thickness in a rangebounded by any of the preceding values. Thickness is determined bycomparison of particle sizes of the coated particle and thecorresponding uncoated core particle using dynamic light scattering.

In some embodiments, two or more surface-altering agents, such as two ormore of a PEG-PPO-PEG triblock copolymer, a synthetic polymer havingpendant OH groups (e.g. PVA), and a polysorbate, may be present in thecoating. Furthermore, although many of the embodiments described hereininvolve a single coating, in other embodiments, a particle may includemore than one coating (e.g., at least two, three, four, five, or morecoatings), and each coating need not be formed of, or comprise, a mucuspenetrating material. In some cases, an intermediate coating (i.e., acoating between the core surface and an outer coating) may include apolymer that facilitates attachment of an outer coating to the coresurface. In many embodiments, an outer coating of a particle includes apolymer comprising a material that facilitates the transport of theparticle through mucus.

Pharmaceutical Formulations

A subject composition may optionally comprise ophthalmically acceptablecarriers, additives, diluents, or a combination thereof. For ophthalmicapplication, solutions or medicaments may be prepared using aphysiological saline solution as a carrier or diluent. Ophthalmicsolutions may be maintained at a physiologic pH with an appropriatebuffer system. The formulations may also contain conventional additives,such as pharmaceutically acceptable buffers, preservatives, stabilizersand surfactants.

Pharmaceutical compositions described herein and for use in accordancewith the articles and methods described herein may include apharmaceutically acceptable excipient or carrier. A pharmaceuticallyacceptable excipient or pharmaceutically acceptable carrier may includea non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any suitable type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose, and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose, andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; detergents such as Tween 80; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator. As would be appreciated by one of skill in this art, theexcipients may be chosen based on the route of administration asdescribed below, the pharmaceutical agent being delivered, time courseof delivery of the agent, etc.

A subject composition may include one or more buffers. Examples include,but are not limited to, acetate buffers, citrate buffers, phosphatebuffers, borate buffers, lactate buffers, NaOH/Trolamine buffers, or acombination thereof such as phosphate and citrate or borate and citrate.Acids or bases, such as HCl and NaOH, may be used to adjust the pH ofthese formulations as needed. The amount of buffer used may vary. Insome embodiments, the buffer may have a concentration in a range ofabout 1 nM to about 100 mM.

A subject composition may include one or more preservatives. Thepreservatives may vary, and may include any compound or substancesuitable for reducing or preventing microbial contamination in anophthalmic liquid subject to multiple uses from the same container.Preservatives that may be used in the pharmaceutical compositionsdisclosed herein include, but are not limited to, cationic preservativessuch as quaternary ammonium compounds including benzalkonium chloride,polyquaternium-1 (Polyquad®), and the like; guanidine-basedpreservatives including PHMB, chlorhexidine, and the like;chlorobutanol; mercury preservatives such as thimerosal, phenylmercuricacetate and phenylmercuric nitrate; and other preservatives such asbenzyl alcohol. In some embodiments, a preservative may have aconcentration of about 10 ppm to about 200 ppm, about 10 ppm to about300 ppm, or about 50 ppm to about 150 ppm.

A subject composition may include one or more surfactants of thefollowing classes: alcohols; amine oxides; block polymers; carboxylatedalcohol or alkylphenol ethoxylates; carboxylic acids/fatty acids;ethoxylated alcohols; ethoxylated alkylphenols; ethoxylated arylphenols; ethoxylated fatty acids; ethoxylated; fatty esters or oils(animal & veg.); fatty esters; fatty acid methyl ester ethoxylates;glycerol esters; glycol esters; lanolin-based derivatives; lecithin andlecithin derivatives; lignin and lignin derivatives; methyl esters;monoglycerides and derivatives; polyethylene glycols; polymericsurfactants; propoxylated & ethoxylated fatty acids, alcohols, or alkylphenols; protein-based surfactants; sarcosine derivatives; sorbitanderivatives; sucrose and glucose esters and derivatives. The amount ofsurfactant may vary. In some embodiments, the amount of any surfactantsuch as those listed above may be about 0.001 to about 5%, about 0.1% toabout 2%, or about 0.1% to about 1%.

A subject composition may include one or more tonicity adjusters. Thetonicity adjusters may vary, and may include any compound or substanceuseful for adjusting the tonicity of an ophthalmic liquid. Examplesinclude, but are not limited to, salts, particularly sodium chloride orpotassium chloride, organic compounds such as propylene glycol,mannitol, or glycerin, or any other suitable ophthalmically acceptabletonicity adjustor. The amount of tonicity adjuster may vary dependingupon whether an isotonic, hypertonic, or hypotonic liquid is desired. Insome embodiments, the amount of a tonicity agent such as those listedabove may be at least about 0.0001% up to about 1%, about 2%, or about5%. In some embodiments a subject composition comprises glycerin.

The osmolality of a subject composition may be hypotonic, isotonic, orhypertonic. For example, a subject composition may have an osmolarity ofabout 200-250 mOsm/kg, about 250-280 mOsm/kg, about 280-320 mOsm/kg,about 290-310 mOsm/kg, about 295-305 mOsm/kg, about 300 mOsm/kg(isotonic), about 300-350 mOsm/kg, or any osmolarity in a range boundedby any of these values. To achieve a formulation of an osmolarity ofabout 300 mOsm/kg, the concentration of sodium chloride in theformulation is typically about 0.9%. A combination of 1.2% glycerin and0.45% sodium chloride generally also yields an isotonic solution.

A subject composition may include an antioxidant such as sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole, and butylated hydroxytoluene.

A subject composition may include a chelating agent such as edetatedisodium.

A subject composition may be suitable for administration to an eye, suchas topical administration to the eye or direct injection into the eye.

Generally, it is desirable for a drug to be pure. For example, it shouldcontain low levels of impurities, such as degradants formed duringsterilization or other processing steps, or formed over time duringstorage. In some embodiments, the level of any degradant of thepharmaceutical agent, such as a hydrocortisone derivative disclosedherein, is no more than about 1 wt %, about 0.9 wt %, about 0.8 wt %,about 0.7 wt %, about 0.6 wt %, about 0.5 wt %, about 0.4 wt %, about0.3 wt %, about 0.2 wt %, about 0.15 wt %, about 0.1 wt %, about 0.03 wt%, about 0.01 wt %, about 0.003 wt %, or about 0.001 wt % relative tothe weight of the pharmaceutical agent.

A subject composition may be administered by any suitable route, such asorally in any acceptable form (e.g., tablet, liquid, capsule, powder,and the like); topically in any acceptable form (e.g., patch, eye drops,creams, gels, nebulization, punctal plug, drug eluting contact,iontophoresis, and ointments); by injection in any acceptable form(e.g., periocular, intravenous, intraperitoneal, intramuscular,subcutaneous, parenteral, and epidural); by inhalation; and by implantor the use of reservoirs (e.g., subcutaneous pump, intrathecal pump,suppository, biodegradable delivery system, non-biodegradable deliverysystem and other implanted extended or slow release device orformulation). The target may be the eye or another organ or tissue. Insome embodiments, a subject composition is administered to an eye inorder to deliver the pharmaceutical agent to a tissue in the eye of thesubject.

A subject composition may be administered at any suitable frequency. Forexample, two or more doses of a subject composition may be administeredto subject, e.g. to an eye of a subject, wherein the period betweenconsecutive doses is at least about 4 hours, at least about 6 hours, atleast about 8 hours, at least about 12 hours, at least about 24 hours,at least about 36 hours, or at least about 48 hours, at least a week, orat least a month.

A subject composition may be administered to treat, diagnose, prevent,or manage a disease or condition in a subject, including a human beingor a non-human animal, such as a mammal. In some embodiments, thecondition is an ocular condition, such as condition affecting theanterior or front of the eye, such as post-surgical inflammation,uveitis, infections, aphakia, pseudophakia, astigmatism, blepharospasm,cataract, conjunctival diseases, conjunctivitis, corneal diseases,corneal ulcer, dry eye syndromes, eyelid diseases, lacrimal apparatusdiseases, lacrimal duct obstruction, myopia, presbyopia; pupildisorders, corneal neovascularization; refractive disorders, andstrabismus. Glaucoma can be considered to be a front of the eye ocularcondition in some embodiments because a clinical goal of glaucomatreatment can be to reduce a hypertension of aqueous fluid in theanterior chamber of the eye (i.e., reduce intraocular pressure).

The leading causes of vision impairment and blindness are conditionslinked to the posterior segment of the eye. These conditions mayinclude, without limitation, age-related ocular degenerative diseasessuch as, macular degeneration, including acute macular degeneration,exudative and non-exudative age related macular degeneration(collectively AMD), proliferative vitreoretinopathy (PVR), retinalocular condition, retinal damage, macular edema (e.g., cystoid macularedema (CME) or (diabetic macular edema (DME)), endophthalmitis;intraocular melanoma; acute macular neuroretinopathy; Behcet's disease;choroidal neovascularization; uveitis; diabetic uveitis; histoplasmosis;infections, such as fungal or viral-caused infections; edema; multifocalchoroiditis; ocular trauma which affects a posterior ocular site orlocation; ocular tumors; retinal disorders, such as central retinal veinocclusion, diabetic retinopathy (including proliferative diabeticretinopathy), retinal arterial occlusive disease, retinal detachment,uveitic retinal disease; sympathetic opthalmia; Vogt Koyanagi-Harada(VKH) syndrome; uveal diffusion; a posterior ocular condition caused byor influenced by an ocular laser treatment; posterior ocular conditionscaused by or influenced by a photodynamic therapy, photocoagulation,radiation retinopathy, epiretinal membrane disorders, branch retinalvein occlusion, anterior ischemic optic neuropathy, non-retinopathydiabetic retinal dysfunction, retinitis pigmentosa, retinoblastoma.Glaucoma can be considered a posterior ocular condition in someembodiments because the therapeutic goal is to prevent the loss of orreduce the occurrence of loss of vision due to damage to or loss ofretinal cells or optic nerve cells (i.e., neuroprotection). In fact,certain forms of glaucoma are not characterized by high IOP, but mainlyby retinal degeneration alone.

Some embodiments include administering a subject composition to treatinflammation, macular degeneration, macular edema, uveitis, dry eye, orglaucoma.

Preparation of Coated Particles

While there are many potential ways to coat drug or core particles witha surface-altering agent, typically this could involve milling theparticles (such as drug particles) with a surface-altering agent orincubating particles in an aqueous solution in the presence of asurface-altering agent. Another useful method involves dissolving a drugin an organic solvent and emulsifying the solution in water using thesurface-altering agent as a surfactant, then removing the organicsolvent by evaporation (e.g. by rotary evaporation). Combinations ofthese methods may also be used.

In a wet milling process, milling can be performed in a dispersion(e.g., an aqueous dispersion) containing one or more surface-alteringagents, a grinding medium, a solid to be milled (e.g., a solidpharmaceutical agent), and a solvent. Any suitable amount of asurface-altering agent can be included in the solvent. In someembodiments, a surface-altering agent may be present in the solvent inan amount of at least about 0.001% (wt % or weight to volume (w:v)), atleast about 0.01%, at least about 0.1%, at least about 0.5%, at leastabout 1%, at least about 2%, at least about 3%, at least about 4%, atleast about 5%, at least about 6%, at least about 7%, at least about 8%,at least about 10%, at least about 12%, at least about 15%, at leastabout 20%, at least about 40%, at least about 60%, or at least about 80%of the solvent. In some cases, the surface-altering agent may be presentin the solvent in an amount of about 100% (e.g., in an instance wherethe surface-altering agent is the solvent). In other embodiments, thesurface-altering agent may be present in the solvent in an amount ofless than or equal to about 100%, less than or equal to about 80%, lessthan or equal to about 60%, less than or equal to about 40%, less thanor equal to about 20%, less than or equal to about 15%, less than orequal to about 12%, less than or equal to about 10%, less than or equalto about 8%, less than or equal to about 7%, less than or equal to about6%, less than or equal to about 5%, less than or equal to about 4%, lessthan or equal to about 3%, less than or equal to about 2%, or less thanor equal to about 1% of the solvent. Combinations of theabove-referenced ranges are also possible (e.g., an amount of less thanor equal to about 5% and at least about 1% of the solvent). Other rangesare also possible. In certain embodiments, the surface-altering agent ispresent in the solvent in an amount of about 0.01-2% of the solvent. Incertain embodiments, the surface-altering agent is present in thesolvent in an amount of about 0.2-20% of the solvent. In certainembodiments, the surface-altering agent is present in the solvent in anamount of about 0.1% of the solvent. In certain embodiments, thesurface-altering agent is present in the solvent in an amount of about0.4% of the solvent. In certain embodiments, the surface-altering agentis present in the solvent in an amount of about 1% of the solvent. Incertain embodiments, the surface-altering agent is present in thesolvent in an amount of about 2% of the solvent. In certain embodiments,the surface-altering agent is present in the solvent in an amount ofabout 5% of the solvent. In certain embodiments, the surface-alteringagent is present in the solvent in an amount of about 10% of thesolvent.

The particular range chosen may influence factors that may affect theability of the particles to penetrate mucus such as the stability of thecoating of the surface-altering agent on the particle surface, theaverage thickness of the coating of the surface-altering agent on theparticles, the orientation of the surface-altering agent on theparticles, the density of the surface altering agent on the particles,surface-altering agent:drug ratio, drug concentration, the size,dispersibility, and polydispersity of the particles formed, and themorphology of the particles formed.

The pharmaceutical agent may be present in the solvent in any suitableamount. In some embodiments, the pharmaceutical agent is present in anamount of at least about 0.001% (wt % or % weight to volume (w:v)), atleast about 0.01%, at least about 0.1%, at least about 0.5%, at leastabout 1%, at least about 2%, at least about 3%, at least about 4%, atleast about 5%, at least about 6%, at least about 7%, at least about 8%,at least about 10%, at least about 12%, at least about 15%, at leastabout 20%, at least about 40%, at least about 60%, or at least about 80%of the solvent. In some cases, the pharmaceutical agent may be presentin the solvent in an amount of less than or equal to about 100%, lessthan or equal to about 90%, less than or equal to about 80%, less thanor equal to about 60%, less than or equal to about 40%, less than orequal to about 20%, less than or equal to about 15%, less than or equalto about 12%, less than or equal to about 10%, less than or equal toabout 8%, less than or equal to about 7%, less than or equal to about6%, less than or equal to about 5%, less than or equal to about 4%, lessthan or equal to about 3%, less than or equal to about 2%, or less thanor equal to about 1% of the solvent. Combinations of theabove-referenced ranges are also possible (e.g., an amount of less thanor equal to about 20% and at least about 1% of the solvent). In someembodiments, the pharmaceutical agent is present in the above ranges butin w:v

The ratio of surface-altering agent to pharmaceutical agent in a solventmay also vary. In some embodiments, the ratio of surface-altering agentto pharmaceutical agent may be at least 0.001:1 (weight ratio, molarratio, or w:v ratio), at least 0.01:1, at least 0.01:1, at least 1:1, atleast 2:1, at least 3:1, at least 5:1, at least 10:1, at least 25:1, atleast 50:1, at least 100:1, or at least 500:1. In some cases, the ratioof surface-altering agent to pharmaceutical agent may be less than orequal to 1000:1 (weight ratio or molar ratio), less than or equal to500:1, less than or equal to 100:1, less than or equal to 75:1, lessthan or equal to 50:1, less than or equal to 25:1, less than or equal to10:1, less than or equal to 5:1, less than or equal to 3:1, less than orequal to 2:1, less than or equal to 1:1, or less than or equal to 0.1:1.Combinations of the above-referenced ranges are possible (e.g., a ratioof at least 5:1 and less than or equal to 50:1). Other ranges are alsopossible.

It should be appreciated that while in some embodiments the stabilizerused for milling forms a coating on a particle surface, which coatingrenders particle mucus penetrating, in other embodiments, the stabilizermay be exchanged with one or more other surface-altering agents afterthe particle has been formed. For example, in one set of methods, afirst stabilizer/surface-altering agent may be used during a millingprocess and may coat a surface of a core particle, and then all orportions of the first stabilizer/surface-altering agent may be exchangedwith a second stabilizer/surface-altering agent to coat all or portionsof the core particle surface. In some cases, the secondstabilizer/surface-altering agent may render the particle mucuspenetrating more than the first stabilizer/surface-altering agent. Insome embodiments, a core particle having a coating including multiplesurface-altering agents may be formed.

Any suitable grinding medium can be used for milling. In someembodiments, a ceramic and/or polymeric material and/or a metal can beused. Examples of suitable materials may include zirconium oxide,silicon carbide, silicon oxide, silicon nitride, zirconium silicate,yttrium oxide, glass, alumina, alpha-alumina, aluminum oxide,polystyrene, poly(methyl methacrylate), titanium, steel. A grindingmedium may have any suitable size. For example, the grinding medium mayhave an average diameter of at least about 0.1 mm, at least about 0.2mm, at least about 0.5 mm, at least about 0.8 mm, at least about 1 mm,at least about 2 mm, or at least about 5 mm. In some cases, the grindingmedium may have an average diameter of less than or equal to about 5 mm,less than or equal to about 2 mm, less than or equal to about 1 mm, lessthan or equal to about 0.8, less than or equal to about 0.5 mm, or lessthan or equal to about 0.2 mm. Combinations of the above-referencedranges are also possible (e.g., an average diameter of at least about0.5 millimeters and less than or equal to about 1 mm). Other ranges arealso possible.

Any suitable solvent may be used for milling. The choice of solvent maydepend on factors such as the solid material (e.g., pharmaceuticalagent) being milled, the particular type of stabilizer/surface-alteringagent being used (e.g., one that may render the particle mucuspenetrating), the grinding material be used, among other factors.Suitable solvents may be ones that do not substantially dissolve thesolid material or the grinding material, but dissolve thestabilizer/surface-altering agent to a suitable degree. Non-limitingexamples of solvents may include water, buffered solutions, otheraqueous solutions, alcohols (e.g., ethanol, methanol, butanol), andmixtures thereof that may optionally include other components such aspharmaceutical excipients, polymers, pharmaceutical agents, salts,preservative agents, viscosity modifiers, tonicity modifier, tastemasking agents, antioxidants, pH modifier, and other pharmaceuticalexcipients. In other embodiments, an organic solvent can be used.

The following embodiments are contemplated:

Embodiment 1

A pharmaceutical composition suitable for administration to an eye,comprising: a plurality of coated particles, comprising: a core particlecomprising a hydrocortisone derivative is

and a mucus penetration-enhancing coating comprising a surface-alteringagent surrounding the core particle, wherein the surface-altering agentcomprises one or more of the following components: a) a triblockcopolymer comprising a hydrophilic block-hydrophobic block-hydrophilicblock configuration, wherein the hydrophobic block has a molecularweight of at least about 2 kDa, and the hydrophilic blocks constitute atleast about 15 wt % of the triblock copolymer, wherein the hydrophobicblock associates with the surface of the core particle, and wherein thehydrophilic block is present at the surface of the coated particle andrenders the coated particle hydrophilic, b) a synthetic polymer havingpendant hydroxyl groups on the backbone of the polymer, the polymerhaving a molecular weight of at least about 1 kDa and less than or equalto about 1000 kDa, wherein the polymer is at least about 30% hydrolyzedand less than about 95% hydrolyzed, or c) a polysorbate, wherein thesurface altering agent is present on the outer surface of the coreparticle at a density of at least 0.01 molecules/nm², wherein thesurface altering agent is present in the pharmaceutical composition inan amount of between about 0.001% to about 5% by weight; and anophthalmically acceptable carrier, additive, or diluent.

Embodiment 2

A pharmaceutical composition suitable for treating an ocular disorder byadministration to an eye, comprising: a plurality of coated particles,comprising: a core particle comprising a hydrocortisone derivativeselected from Compounds 1, 2, and 3, and a mucus penetration-enhancingcoating comprising a surface-altering agent surrounding the coreparticle, wherein the surface-altering agent comprises one or more ofthe following components: a) a triblock copolymer comprising ahydrophilic block-hydrophobic block-hydrophilic block configuration,wherein the hydrophobic block has a molecular weight of at least about 2kDa, and the hydrophilic blocks constitute at least about 15 wt % of thetriblock copolymer, b) a synthetic polymer having pendant hydroxylgroups on the backbone of the polymer, the polymer having a molecularweight of at least about 1 kDa and less than or equal to about 1000 kDa,wherein the polymer is at least about 30% hydrolyzed and less than about95% hydrolyzed, or c) a polysorbate, wherein the plurality of coatedparticles have an average smallest cross-sectional dimension of lessthan about 1 micron; and wherein the coating on the core particle ispresent in a sufficient amount to increase the concentration of thehydrocortisone derivative and/or hydrocortisone metabolite in a corneaor an aqueous humor after administration when administered to the eye,compared to the concentration of the hydrocortisone derivative in thecornea or the aqueous humor when administered as a core particle withoutthe coating.

Embodiment 3

The pharmaceutical composition according to embodiments 1 or 2 whereinthe hydrocortisone derivative is(10R,11S,13S,17R)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl3-(phenylsulfonyl)propanoate.

Embodiment 4

The pharmaceutical composition according to embodiments 1 or 2 whereinthe hydrocortisone derivative is(10R,11S,13S,17R)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-ylfuran-2-carboxylate

Embodiment 5

The pharmaceutical composition according to embodiments 1 or 2 whereinthe hydrocortisone derivative is(10R,11S,13S,17R)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl2-(4-bromophenyl)acetate.

Embodiment 6

The pharmaceutical composition of embodiment 1 or 2, wherein thehydrocortisone derivative is Compound 1:

Embodiment 7

The pharmaceutical composition of embodiment 6, wherein Compound 1 is incrystalline form B having XRPD peaks at 5.88, 10.36, 13.18, 14.40,15.55, 17.57, and 20.82±0.2° 2θ.

Embodiment 8

The pharmaceutical composition of embodiment 1 or 2, wherein thehydrocortisone derivative is Compound 2:

Embodiment 9

The pharmaceutical composition of embodiment 8, wherein Compound 2 is incrystalline form A having XRPD peaks at 5.83, 10.09, 11.72, 14.49,15.32, and 15.66±0.2° 2θ.

Embodiment 10

The pharmaceutical composition of embodiment 1 or 2, wherein thehydrocortisone derivative is Compound 3:

Embodiment 11

The pharmaceutical composition of claim 10, wherein Compound 3 is incrystalline form A having XRPD peaks at 5.08, 7.18, 13.90, and20.45±0.2° 2θ.

Embodiment 12

The pharmaceutical composition of claim 10, wherein Compound 3 is incrystalline form B having XRPD peaks at 8.88, 12.66, 14.34, 19.02,20.28, 20.63 and 25.71±0.2° 2θ.

Embodiment 13

The pharmaceutical composition of any one of embodiments 1-12, whereinthe surface-altering agent is covalently attached to the core particles.

Embodiment 14

The pharmaceutical composition of any one of embodiments 1-12, whereinthe surface-altering agent is non-covalently adsorbed to the coreparticles.

Embodiment 15

The pharmaceutical composition of any one of embodiments 1-14, whereinthe surface-altering agent is present on the surfaces of the coatedparticles at a density of at least about 0.1 molecules per nanometersquared.

Embodiment 16

The pharmaceutical composition of any one of embodiments 1-12, whereinthe surface-altering agent comprises the triblock copolymer.

Embodiment 17

The pharmaceutical composition of any one of embodiments 1-12, whereinthe surface-altering agent comprises the triblock copolymer, wherein thehydrophilic blocks of the triblock copolymer constitute at least about30 wt % of the triblock polymer and less than or equal to about 80 wt %of the triblock copolymer.

Embodiment 18

The pharmaceutical composition of embodiment 16 or 17, wherein thehydrophobic block portion of the triblock copolymer has a molecularweight of about 3 kDa to about 8 kDa.

Embodiment 19

The pharmaceutical composition of any one of embodiments 16-18, whereinthe triblock copolymer is poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide).

Embodiment 20

The pharmaceutical composition of any one of embodiments 1-12, whereinthe surface-altering agent comprises a linear polymer having pendanthydroxyl groups on the backbone of the polymer.

Embodiment 21

The pharmaceutical composition of any one of embodiments 1-20, whereinthe surface-altering agent has a molecular weight of at least about 4kDa.

Embodiment 22

The pharmaceutical composition of any one of embodiments 1-12, whereinthe surface altering agent is polyvinyl alcohol.

Embodiment 23

The pharmaceutical composition of embodiment 22, wherein the poly(vinylalcohol) is about 70% to about 94% hydrolyzed.

Embodiment 24

The pharmaceutical composition of any one of embodiments 1-23, whereinthe hydrocortisone derivative is crystalline.

Embodiment 25

The pharmaceutical composition of any one of embodiments 1-23, whereinthe hydrocortisone derivative is amorphous.

Embodiment 26

The pharmaceutical composition of any one of embodiments 1-25, whereinthe core particles comprise a hydrocortisone derivative that isencapsulated in a polymer, a lipid, a protein, or a combination thereof.

Embodiment 27

The pharmaceutical composition of any one of embodiments 1-26, whereinthe hydrocortisone derivative constitutes at least about 80 wt % of thecore particle.

Embodiment 28

The pharmaceutical composition of any one of embodiments 1-27, whereinthe coated particles have an average size of about 10 nm to about 1 μm.

Embodiment 29

The pharmaceutical composition of any one of embodiments 1-28,comprising one or more degradants of the hydrocortisone derivative, andwherein the concentration of each degradant is 0.1 wt % or less relativeto the weight of the hydrocortisone.

Embodiment 30

The pharmaceutical composition of any one of embodiments 1-29, whereinthe polydispersity index of the composition is less than or equal toabout 0.5.

Embodiment 31

The pharmaceutical composition of any one of embodiments 1-30, whereinthe pharmaceutical composition is suitable for topical administration tothe eye.

Embodiment 32

The pharmaceutical composition of any one of embodiments 1-31, whereinthe pharmaceutical composition is suitable for direct injection into theeye.

Embodiment 33

The pharmaceutical composition of any one of embodiments 1-32, whereinthe ophthalmically acceptable carrier, additive, or diluent comprisesglycerin.

Embodiment 34

A method of treating, diagnosing, preventing, or managing an ocularcondition in a subject, the method comprising: administering apharmaceutical composition of any one of embodiments 1-32 to an eye of asubject and thereby delivering the hydrocortisone derivative and/orhydrocortisone metabolite to a tissue in the eye of the subject.

Embodiment 35

The method of embodiment 34, wherein after administering thepharmaceutical composition topically to the eye, an ophthalmicallyefficacious level of the hydrocortisone derivative and/or itshydrocortisone metabolite are/is delivered to a palpebral conjunctiva, abulbar conjunctiva, a fornix conjunctiva, an aqueous humor, an anteriorsclera, or a cornea for at least 12 hours after administration.

Embodiment 36

The method of any one of embodiments 34 or 35, wherein thehydrocortisone derivative and/or its hydrocortisone metabolite are/isdelivered to a tissue in the front of the eye of the subject.

Embodiment 37

The method of embodiment 34, wherein the hydrocortisone derivativeand/or its hydrocortisone metabolite are/is delivered to a tissue in theback of the eye of the subject.

Embodiment 38

The method of embodiment 34, wherein the tissue is a retina, a macula, aposterior sclera, vitreous humor, or a choroid.

Embodiment 39

The method of any one of embodiments 34-38, wherein the ocular conditionis inflammation, macular degeneration, macular edema, uveitis, glaucoma,or dry eye.

EXAMPLES Example 1

The following describes a non-limiting example of a method of formingnon-polymeric solid particles into mucus-penetrating particles. Pyrene,a hydrophobic naturally fluorescent compound, was used as the coreparticle and was prepared by a milling process in the presence ofvarious surface-altering agents. The surface-altering agents formedcoatings around the core particles. Different surface-altering agentswere evaluated to determine effectiveness of the coated particles inpenetrating mucus.

Pyrene was milled in aqueous dispersions in the presence of varioussurface-altering agents to determine whether certain surface-alteringagents can: 1) aid particle size reduction to several hundreds ofnanometers and 2) physically (non-covalently) coat the surface ofgenerated nanoparticles with a coating that would minimize particleinteractions with mucus constituents and prevent mucus adhesion. Thesurface-altering agents tested included a variety of polymers,oligomers, and small molecules listed in Table 3 below, includingpharmaceutically relevant excipients such as poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers(Pluronic® copolymers), polyvinylpyrrolidones (Kollidon), andhydroxypropyl methylcellulose (Methocel), etc.

TABLE 3 Surface-altering agents tested with pyrene as a model compound.Acronym or Stabilizer Trade Name Grade or Molecular Weight Polymericsurface-altering agents Poly(ethylene oxide)- Pluronic ® F127, F108,F68, F87, F38, P123, poly(propylene oxide)- P105, P103, P65, L121, L101,L81, poly(ethylene oxide) block L44, L31 copolymers PolyvinylpyrrolidonePVP Kollidon 17 (9K), Kollidon 25 (26K), Kollindon 30 (43K)PVA-poly(ethylene glycol) graft- Kollicoat IR copolymer Hydroxypropylmethylcellulose HPMC Methocel E50, Methocel K100 Oligomericsurface-altering agents Tween 20 Tween 80 Solutol HS 15 Triton X100Tyloxapol Cremophor RH 40 Small molecule surface-altering agents Span 20Span 80 Octyl glucoside Cetytrimethylammonium bromide (CTAB) Sodiumdodecyl sulfate (SDS)

An aqueous dispersion containing pyrene and one of the surface-alteringagents listed above was milled with milling media until particle sizewas reduced below 500 nm. Table 4 lists particle size characteristics ofpyrene particles obtained by milling in the presence of the varioussurface-altering agents. Particle size was measured by dynamic lightscattering. When Pluronic® L101, L81, L44, L31, Span 20, Span 80, orOctyl glucoside were used as surface-altering agents, stablenanosuspensions could not be obtained. Therefore, these surface-alteringagents were excluded from further investigation due to their inabilityto effectively aid particle size reduction.

TABLE 4 Particle size measured by DLS in nanosuspensions obtained bymilling of pyrene with various surface-altering agents. StabilizerN-Ave. D (nm) Pluronic ® F127 239 Pluronic ® F108 267 Pluronic ® P105303 Pluronic ® P103 319 Pluronic ® P123 348 Pluronic ® L121 418Pluronic ® F68 353 Pluronic ® P65 329 Pluronic ® F87 342 Pluronic ® F38298 Pluronic ® L101 not measurable* Pluronic ® L81 not measurable*Pluronic ® L44 not measurable* Pluronic ® L31 not measurable* PVA 13K314 PVA 31K 220 PVA 85K 236 Kollicoat IR 192 Kollidon 17 (PVP 9K) 163Kollidon 25 (PVP 26K) 210 Kollindon 30 (PVP 43K) 185 Methocel E50 160Methocel K100 216 Tween 20 381 Tween 80 322 Solutol HS 378 Triton X100305 Tyloxapol 234 Cremophor RH40 373 SDS 377 CTAB 354 Span 20 notmeasurable* Span 80 not measurable* Octyl glucoside not measurable**milling with Pluronic ® L101, L81, L44, L31, Span 20, Span 80, Octylglucoside failed to effectively reduce pyrene particle size and producestable nanosuspensions.

The mobility and distribution of pyrene nanoparticles from the producednanosuspensions in human cervicovaginal mucus (CVM) were characterizedusing fluorescence microscopy and multiple particle tracking software.In a typical experiment, ≤0.5 uL of a nanosuspension (diluted ifnecessary to the surfactant concentration of ˜1%) was added to 20 μl offresh CVM along with controls. Conventional nanoparticles (200 nmyellow-green fluorescent carbon/late-modified polystyrene microspheresfrom Invitrogen) were used as a negative control to confirm the barrierproperties of the CVM samples. Red fluorescent polystyrene nanoparticlescovalently coated with PEG 5 kDa were used as a positive control withwell-established MPP behavior. Using a fluorescent microscope equippedwith a CCD camera, 15 s movies were captured at a temporal resolution of66.7 ms (15 frames/s) under 100× magnification from several areas withineach sample for each type of particles: sample (pyrene), negativecontrol, and positive control (natural blue fluorescence of pyreneallowed observing of pyrene nanoparticles separately from the controls).Next, using an image processing software, individual trajectories ofmultiple particles were measured over a time-scale of at least 3.335 s(50 frames). Resulting transport data are presented here in the form oftrajectory-mean velocity V_(mean), i.e., velocity of an individualparticle averaged over its trajectory, and ensemble-average velocity<V_(mean)>, i.e., V_(mean) averaged over an ensemble of particles. Toenable easy comparison between different samples and normalize velocitydata with respect to natural variability in penetrability of CVMsamples, relative sample velocity <V_(mean)>_(rel), was determinedaccording to the formula shown in Equation 1.

$\begin{matrix}{{\text{<}V_{mean}\text{>}_{rel}} = \frac{{\text{<}V_{mean}\text{>}_{Sample}} - {\text{<}V_{mean}\text{>}_{{Negative}\mspace{14mu} {control}}}}{{\text{<}V_{mean}\text{>}_{{Positive}\mspace{14mu} {control}}} - {\text{<}V_{mean}\text{>}_{{Negative}\mspace{14mu} {control}}}}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

Prior to quantifying mobility of the produced pyrene nanoparticles,their spatial distribution in the mucus sample was assessed bymicroscopy at low magnifications (10×, 40×). It was found thatpyrene/Methocel nanosuspensions did not achieve uniform distribution inCVM and strongly aggregated into domains much larger than the mucus meshsize (data not shown). Such aggregation is indicative of mucoadhesivebehavior and effectively prevents mucus penetration. Therefore, furtherquantitative analysis of particle mobility was deemed unnecessary.Similarly to the positive control, all other tested pyrene/stabilizersystems achieved a fairly uniform distribution in CVM. Multiple particletracking confirmed that in all tested samples, the negative controlswere highly constrained, while the positive controls were highly mobileas demonstrated by <V_(mean)> for the positive controls beingsignificantly greater than those for the negative controls (Table 5).

TABLE 5 Ensemble-average velocity <V_(mean)> (um/s) and relative samplevelocity <V_(mean)>_(rel) for pyrene/stabilizer nanoparticles (sample)and controls in CVM. Negative Positive Sample Control Control Sample(relative) Stabilizer <V_(mean)> SD <V_(mean)> SD <V_(mean)> SD<V_(mean)>_(rel) SD Pluronic ® F127 0.58 0.18 5.97 0.54 6.25 0.72 1.050.18 Pluronic ® F108 0.43 0.64 5.04 1.88 4.99 1.47 0.99 0.55 Pluronic ®P105 0.56 0.52 4.38 1.36 4.47 2.11 1.02 0.69 Pluronic ® P103 0.58 0.774.5 2.01 4.24 1.95 0.93 0.74 Pluronic ® P123 0.56 0.44 4.56 1.44 3.991.66 0.86 0.54 Pluronic ® L121 0.42 0.3 4.27 2.04 0.81 0.51 0.10 0.16Pluronic ® F68 0.56 0.52 4.38 1.36 0.81 0.7 0.07 0.23 Pluronic ® P650.26 0.25 4.52 2.15 0.53 0.56 0.06 0.15 Pluronic ® F87 0.95 1.6 5.061.34 0.74 0.78 −0.05 −0.43 Pluronic ® F38 0.26 0.1 5.73 0.84 0.54 0.290.05 0.06 Kollicoat IR 0.62 0.62 5.39 0.55 0.92 0.81 0.06 0.22 Kollidon17 1.69 1.8 5.43 0.98 0.82 0.59 −0.23 −0.52 Kollidon 25 0.41 0.34 5.040.64 1.29 1.09 0.19 0.25 Kollindon 30 0.4 0.2 4.28 0.57 0.35 0.11 −0.010.06 Methocel E50** Methocel K100** Tween 20 0.77 0.93 5.35 1.76 1.582.02 0.18 0.49 Tween 80 0.46 0.34 3.35 1.89 0.94 0.5 0.17 0.24 SolutolHS 0.42 0.13 3.49 0.5 0.8 0.6 0.12 0.20 Triton X100 0.26 0.13 4.06 1.110.61 0.19 0.09 0.07 Tyloxapol 0.5 0.5 3.94 0.58 0.42 0.23 −0.02 −0.16Cremophor RH40 0.48 0.21 3.2 0.97 0.49 0.24 0.00 0.12 SDS 0.3 0.12 5.990.84 0.34 0.15 0.01 0.03 CTAB 0.39 0.09 4.75 1.79 0.32 0.31 −0.02 −0.07*Did not produce stable nanosuspensions, hence not mucus-penetrating(velocity in CVM not measured) **Aggregated in CVM, hence notmucus-penetrating (velocity in CVM not measured)

It was discovered that nanoparticles obtained in the presence of certainsurface-altering agents migrate through CVM at the same rate or nearlythe same velocity as the positive control. Specifically, pyrenenanoparticles stabilized with Pluronic® F127, F108, P123, P105, and P103exhibited <V_(mean)> that exceeded those of the negative controls byapproximately an order of magnitude and were indistinguishable, withinexperimental error, from those of the positive controls, as shown inTable 5 and FIG. 2A. For these samples, <V_(mean)>_(rel) values exceeded0.5, as shown in FIG. 2B.

FIGS. 3A-3D are histograms showing distribution of V_(mean) within anensemble of particles. These histograms illustrate muco-diffusivebehavior of samples stabilized with Pluronic® F127 and Pluronic® F108(similar histograms were obtained for samples stabilized with Pluronic®P123, P105, and P103, but are not shown here) as opposed tomuco-adhesive behavior of samples stabilized with Pluronic® 87, andKollidon 25 (chosen as representative muco-adhesive samples).

To identify the characteristics of Pluronic® copolymers that renderpyrene nanoparticles mucus penetrating, <V_(mean)>_(rel) of thepyrene/Pluronic® nanoparticles was mapped with respect to molecularweight of the PPO block and the PEO weight content (%) of the Pluronic®copolymers used (FIG. 4). It was concluded that at least those Pluronic®copolymers that have the PPO block of at least 3 kDa and the PEO contentof at least about 30 wt % rendered the nanoparticles mucus-penetrating.

Example 2

The following describes a non-limiting example of a method of formingmucus-penetrating particles from pre-fabricated polymeric particles byphysical adsorption of certain poly(vinyl alcohol) polymers (PVA).Carboxylated polystyrene nanoparticles (PSCOO) were used as theprefabricated particle/core particle with a well-established stronglymucoadhesive behavior. The PVAs acted as surface-altering agents formingcoatings around the core particles. PVA of various molecular weights(MW) and hydrolysis degrees were evaluated to determine effectiveness ofthe coated particles in penetrating mucus.

PSCOO⁻ particles were incubated in aqueous solution in the presence ofvarious PVA polymers to determine whether certain PVAs can physically(non-covalently) coat the core particle with a coating that wouldminimize particle interactions with mucus constituents and lead to rapidparticle penetration in mucus. In these experiments, the PVA acted as acoating around the core particles, and the resulting particles weretested for their mobility in mucus, although in other embodiments, PVAmay be exchanged with other surface-altering agents that can increasemobility of the particles in mucus. The PVAs tested ranged in theaverage molecular weight from 2 kDa to 130 kDa and in the averagehydrolysis degree from 75% to 99+%. The PVAs that were tested are listedin Table 2, shown above.

The particle modification process was as follows: 200 nm red fluorescentPSCOO⁻ were purchased from Invitrogen. The PSCOO⁻ particles (0.4-0.5 wt%) were incubated in an aqueous PVA solution (0.4-0.5 wt %) for at least1 hour at room temperature.

The mobility and distribution of the modified nanoparticles in CVM werecharacterized using fluorescence microscopy and multiple particletracking software in a method similar to that described above. Multipleparticle tracking confirmed that in all tested CVM samples the negativecontrols were constrained, while the positive controls were mobile asdemonstrated by the differences in <V_(mean)> for the positive andnegative controls (Table 6).

TABLE 6 Transport of nanoparticles incubated with various PVA (sample)and controls in CVM: Ensemble-average velocity <V_(mean)> (μm/s) andrelative sample velocity <V_(mean)>_(rel). Negative Positive SampleControl Control Sample (relative) Stabilizer <V_(mean)> SD <V_(mean)> SD<V_(mean)> SD <V_(mean)>_(rel) SD PVA2K75 1.39 0.33 3.3 0.68 3.44 0.71.07 0.59 PVA9K80 0.4 0.08 5.13 1.16 4.88 1.74 0.95 0.44 PVA13K87 0.560.61 5.23 1.24 4.92 1.77 0.93 0.49 PVA31K87 0.53 0.63 4.48 1.38 3.691.94 0.80 0.60 PVA57K86 0.5 0.25 5.74 1.11 4.76 0.91 0.81 0.25 PVA85K870.29 0.28 4.25 0.97 4.01 0.71 0.94 0.31 PVA105K80 0.98 0.52 5.44 0.864.93 0.66 0.89 0.27 PVA130K87 1.41 0.56 3.75 0.82 3.57 0.6 0.92 0.53PVA95K95 0.51 0.36 3.19 0.68 0.45 0.19 −0.02 −0.15 PVA13K98 0.43 0.173.42 1.65 0.5 0.76 0.02 0.26 PVA31K98 0.41 0.23 6.03 1.19 0.26 0.14−0.03 −0.05 PVA85K99 0.28 0.1 4.7 0.82 0.53 0.77 0.06 0.18

It was discovered that nanoparticles incubated in the presence ofcertain PVA transported through CVM at the same rate or nearly the samevelocity as the positive control. Specifically, the particles stabilizedwith PVA2K75, PVA9K80, PVA13K87, PVA31K87, PVA57K86, PVA85K87,PVA105K80, and PVA130K87 exhibited <V_(mean)> that significantlyexceeded those of the negative controls and were indistinguishable,within experimental error, from those of the positive controls. Theresults are shown in Table 6 and FIG. 5A. For these samples,<V_(mean)>_(rel) values exceeded 0.5, as shown in FIG. 5B.

On the other hand, nanoparticles incubated with PVA95K95, PVA13K98,PVA31K98, and PVA85K99 were predominantly or completely immobilized asdemonstrated by respective <V_(mean)>_(rel) values of no greater than0.1 (Table 6 and FIG. 5B).

To identify the characteristics of the PVA that render particles mucuspenetrating, <V_(mean)>_(rel) of the nanoparticles prepared byincubation with the various PVAs was mapped with respect to MW andhydrolysis degree of the PVAs used (FIG. 6). It was concluded that atleast those PVAs that have the hydrolysis degree of less than 95%rendered the nanoparticles mucus-penetrating.

To further confirm the ability of the specific PVA grades to convertmucoadhesive particles into mucus-penetrating particles by physicaladsorption, PSCOO⁻ nanoparticles incubated with the various PVAs weretested using the bulk transport assay. In this method, 20 μL of CVM wascollected in a capillary tube and one end is sealed with clay. The openend of the capillary tube is then submerged in 20 μL of an aqueoussuspension of particles which is 0.5% w/v drug. After the desired time,typically 18 hours, the capillary tube is removed from the suspensionand the outside is wiped clean. The capillary containing the mucussample is placed in an ultracentrifuge tube. Extraction media is addedto the tube and incubated for 1 hour while mixing which removes themucus from the capillary tube and extracts the drug from the mucus. Thesample is then spun to remove mucins and other non-soluble components.The amount of drug in the extracted sample can then be quantified usingHPLC. The results of these experiments are in good agreement with thoseof the microscopy method, showing clear differentiation in transportbetween positive (mucus-penetrating particles) and negative controls(conventional particles). The bulk transport results for PSCOO⁻nanoparticles incubated with the various PVAs are shown in FIG. 7A-B.These results corroborate microscopy/particle tracking findings withPSCOO⁻ nanoparticles incubated with the various PVAs and demonstrate theincubating nanoparticles with partially hydrolyzed PVAs enhances mucuspenetration.

Example 3

The following describes a non-limiting example of a method of formingmucus-penetrating particles by an emulsification process in the presenceof certain poly(vinyl alcohol) polymers (PVA). Polylactide (PLA), abiodegradable pharmaceutically relevant polymer was used as a materialto form the core particle via an oil-in-water emulsification process.The PVAs acted as emulsion surface-altering agents and surface-alteringagents forming coatings around the produced core particles. PVA ofvarious molecular weights (MW) and hydrolysis degrees were evaluated todetermine effectiveness of the formed particles in penetrating mucus.

PLA solution in dichloromethane was emulsified in aqueous solution inthe presence of various PVA to determine whether certain PVAs canphysically (non-covalently) coat the surface of generated nanoparticleswith a coating that would lead to rapid particle penetration in mucus.In these experiments, the PVA acted as a surfactant that forms astabilizing coating around droplets of emulsified organic phase that,upon solidification, form the core particles. The resulting particleswere tested for their mobility in mucus, although in other embodiments,PVA may be exchanged with other surface-altering agents that canincrease mobility of the particles in mucus. The PVAs tested ranged inthe average molecular weight from 2 kDa to 130 kDa and in the averagehydrolysis degree from 75% to 99+%. The PVAs that were tested are listedin Table 2, shown above.

The emulsification-solvent evaporation process was as follows:Approximately 0.5 mL of 20-40 mg/ml solution of PLA (Polylactide grade100DL7A, purchased from Surmodics) in dichloromethane was emulsified inapproximately 4 mL of an aqueous PVA solution (0.5-2 wt %) by sonicationto obtain a stable emulsion with the target number-average particle sizeof <500 nm. Obtained emulsions were immediately subjected to exhaustiverotary evaporation under reduced pressure at room temperature to removethe organic solvent. Obtained suspensions were filtered through 1 micronglass fiber filters to remove any agglomerates. Table 7 lists theparticle size characteristics of the nanosuspensions obtained by thisemulsification procedure with the various PVA. In all cases, afluorescent organic dye Nile Red was added to the emulsified organicphase to fluorescently label the resulting particles.

TABLE 7 Particle size measured by DLS in nanosuspensions obtained by theemulsification process of PLA particles with various PVA. PVA GradeZ-Ave D (nm) N-Ave D (nm) PVA2K75 186 156 PVA10K80 208 173 PVA13K98 245205 PVA31K87 266 214 PVA31K98 245 228 PVA85K87 356 301 PVA85K99 446 277PVA95K95 354 301 PVA105K80 361 300 PVA130K87 293 243

The mobility and distribution of the produced nanoparticles in CVM werecharacterized using fluorescence microscopy and multiple-particletracking software in a manner similar to that described above. Multipleparticle tracking confirmed that in all tested CVM samples the negativecontrols were constrained, while the positive controls were mobile asdemonstrated by the differences in <V_(mean)> for the positive andnegative controls (Table 8).

TABLE 8 Transport of PLA nanoparticles obtained by the emulsificationprocess with various PVAs (sample) and controls in CVM: Ensemble-averagevelocity <V_(mean)> (um/s) and relative sample velocity<V_(mean)>_(rel). Negative Positive Sample Control Control Sample(relative) Stabilizer <V_(mean)> SD <V_(mean)> SD <V_(mean)> SD<V_(mean)>_(rel) SD PVA2K75 0.95 0.64 5.5 0.92 5.51 1.2 1.00 0.39PVA9K80 0.72 0.47 5.61 0.79 4.6 1.5 0.79 0.35 PVA31K87 0.63 0.60 4.941.50 3.36 1.84 0.63 0.51 PVA85K87 0.57 0.4 4.49 1.21 2.9 1.56 0.59 0.45PVA105K80 0.69 0.56 4.85 1.54 3.55 1.26 0.69 0.43 PVA130K87 0.95 0.544.98 1.25 3.46 1.23 0.62 0.39 PVA95K95 1.39 1.28 5.72 1.57 1.63 1.5 0.060.46 PVA13K98 1.02 0.49 5.09 0.99 2.61 1.54 0.39 0.41 PVA31K98 1.09 0.65.09 0.9 2.6 1.13 0.38 0.34 PVA85K99 0.47 0.33 5.04 2.2 0.81 0.77 0.070.19

It was discovered that nanoparticles prepared in the presence of certainPVA transported through CVM at the same rate or nearly the same velocityas the positive control. Specifically, the particles stabilized withPVA2K75, PVA9K80, PVA13K87, PVA31K87, PVA85K87, PVA105K80, and PVA130K87exhibited <V_(mean)> that significantly exceeded those of the negativecontrols and were indistinguishable, within experimental error, fromthose of the positive controls, as shown in Table 8 and FIG. 8A. Forthese samples, <V_(mean)>_(rel) values exceeded 0.5, as shown in FIG.8B.

On the other hand, nanoparticles obtained with PVA95K95, PVA13K98,PVA31K98, and PVA85K99 were predominantly or completely immobilized asdemonstrated by respective <V_(mean)>_(rel) values of no greater than0.4 (Table 8 and FIG. 8B). To identify the characteristics of the PVAthat render particles mucus penetrating, <V_(mean)>_(rel) of thenanoparticles prepared with the various PVAs was mapped with respect toMW and hydrolysis degree of the PVAs used (Table 6 and FIG. 8B). It wasconcluded that at least those PVAs that have the hydrolysis degree ofless than 95% rendered the nanoparticles mucus-penetrating.

Example 4

The following describes a non-limiting example of a method of formingmucus-penetrating non-polymeric solid particles by milling in thepresence of certain poly(vinyl alcohol) polymers (PVA). Pyrene, a modelhydrophobic compound, was used as the core particle processed by amilling. The PVA acted as milling aids facilitating particle sizereduction of the core particles and surface-altering agents formingcoatings around the core particles. PVA of various molecular weights(MW) and hydrolysis degrees were evaluated to determine effectiveness ofthe milled particles in penetrating mucus.

Pyrene was milled in aqueous dispersions in the presence of various PVAto determine whether PVAs of certain MW and hydrolysis degree can: 1)aid particle size reduction to several hundreds of nanometers and 2)physically (non-covalently) coat the surface of generated nanoparticleswith a coating that would minimize particle interactions with mucusconstituents and prevent mucus adhesion. In these experiments, the PVAacted as a coating around the core particles, and the resultingparticles were tested for their mobility in mucus. The PVAs testedranged in the average molecular weight from 2 kDa to 130 kDa and in theaverage hydrolysis degree from 75% to 99+%. The PVAs that were testedare listed in Table 1, shown above. A variety of other polymers,oligomers, and small molecules listed in Table 9, includingpharmaceutically relevant excipients such as polyvinylpyrrolidones(Kollidon), hydroxypropyl methylcellulose (Methocel), Tween, Span, etc.,were tested in a similar manner.

TABLE 9 Other surface-altering agents tested with pyrene as a modelcompound. Chemical Family Grades Polyvinylpyrrolidone (PVP) Kollidon 17Kollidon 25 Kollindon 30 PVA-poly(ethylene glycol) graft-copolymerKollicoat IR Hydroxypropyl methylcellulose (HPMC) Methocel E50 MethocelK100 Non-ionic polyoxyethylene surfactants Solutol HS 15 Span 20 Span 80Triton X100 Tween 20 Tween 80 Tyloxapol Non-ionic small moleculesurfactants Octyl glucoside Ionic small molecule surfactantsCetytrimethylammonium bromide (CTAB) Sodium dodecyl sulfate (SDS)

An aqueous dispersion containing pyrene and one of the surface-alteringagents listed above was stirred with milling media until particle sizewas reduced below 500 nm (as measured by dynamic light scattering).

Table 10 lists particle size characteristics of pyrene particlesobtained by milling in the presence of the various surface-alteringagents. When Span 20, Span 80, or Octyl glucoside was used assurface-altering agents, stable nanosuspensions could not be obtained.Therefore, these surface-altering agents were excluded from furtherinvestigation due to their inability to effectively aid particle sizereduction.

TABLE 10 Particle size measured by DLS in nanosuspensions obtained bymilling of pyrene with various surface-altering agents. Stabilizer Z-AveD (nm) N-Ave D (nm) PVA2K75 340 301 PVA9K80 380 337 PVA13K87 375 326PVA13K98 396 314 PVA31K87 430 373 PVA31K98 344 220 PVA85K87 543 434PVA85K99 381 236 PVA95K95 534 392 PVA130K87 496 450 Kollidon 17 237 163Kollidon 25 307 210 Kollindon 30 255 185 Kollicoat IR 364 192 MethocelE50 244 160 Methocel K100 375 216 Tween 20 567 381 Tween 80 553 322Solutol HS 576 378 Triton X100 410 305 Tyloxapol 334 234 Cremophor RH40404 373 Span 20 not measurable* Span 80 not measurable* Octyl glucosidenot measurable* SDS 603 377 CTAB 432 354 *milling with Span 20, Span 80,Octyl glucoside failed to effectively reduce pyrene particlesize andproduce stable nanosuspensions.

The mobility and distribution of the produced pyrene nanoparticles inCVM were characterized using fluorescence microscopy and multipleparticle tracking software in a manner similar to that described above.Multiple particle tracking confirmed that in all tested CVM samples thenegative controls were constrained, while the positive controls weremobile as demonstrated by the differences in <V_(mean)> for the positiveand negative controls (Table 11).

TABLE 11 Transport of pyrene nanoparticles (sample) obtained withvarious surface-altering agents and controls in CVM: Ensemble-averagevelocity <V_(mean)> (um/s) and relative sample velocity<V_(mean)>_(rel). Negative Positive Sample Control Control Sample(relative) Stabilizer <V_(mean)> SD <V_(mean)> SD <V_(mean)> SD<V_(mean)>_(rel) SD PVA2K75 0.4 0.24 5.73 0.73 4.73 1.08 0.81 0.24PVA9K80 0.36 0.20 6.00 0.70 6.19 1.13 1.03 0.24 PVA13K87 1.01 1.21 5.090.98 4.54 1.03 0.87 0.51 PVA31K87 1.28 1.14 4.88 0.6 4.57 1.123 0.910.55 PVA85K87 1.05 0.9 4.1 0.57 3.3 0.98 0.74 0.51 PVA130K87 0.51 0.825.29 0.73 4.12 1.49 0.76 0.40 PVA95K95 0.4 0.27 4.53 1.03 0.67 0.6 0.070.16 PVA13K98 0.61 0.42 2.13 0.99 1.29 0.57 0.45 0.56 PVA31K98 0.68 0.875.77 1.24 2.69 2.02 0.39 0.45 PVA85K99 0.43 0.23 5.42 0.97 2.23 1.600.36 0.33 Kollicoat IR 0.62 0.62 5.39 0.55 0.92 0.81 0.06 0.22 Kollidon17 1.69 1.8 5.43 0.98 0.82 0.59 −0.23 −0.52 Kollidon 25 0.41 0.34 5.040.64 1.29 1.09 0.19 0.25 Kollindon 30 0.4 0.2 4.28 0.57 0.35 0.11 −0.010.06 Methocel E50* Methocel K100* Tween 20 0.77 0.93 5.35 1.76 1.58 2.020.18 0.49 Tween 80 0.46 0.34 3.35 1.89 0.94 0.5 0.17 0.24 Solutol HS0.42 0.13 3.49 0.5 0.8 0.6 0.12 0.20 Triton X100 0.26 0.13 4.06 1.110.61 0.19 0.09 0.07 Tyloxapol 0.5 0.5 3.94 0.58 0.42 0.23 −0.02 −0.16Cremophor RH40 0.48 0.21 3.2 0.97 0.49 0.24 0.00 0.12 SDS 0.3 0.12 5.990.84 0.34 0.15 0.01 0.03 CTAB 0.39 0.09 4.75 1.79 0.32 0.31 −0.02 −0.07*Aggregated in CVM, hence not mucus-penetrating (velocity in CVM notmeasured)

It was discovered that nanoparticles obtained in the presence of certainexcipients transported through CVM at the same rate or nearly the samevelocity as the positive control. Specifically, pyrene nanoparticlesstabilized with PVA2K75, PVA9K80, PVA13K87, PVA31K87, PVA85K87, andPVA130K87 exhibited <V_(mean)> that significantly exceeded those of thenegative controls and were indistinguishable, within experimental error,from those of the positive controls, as shown in Table 11 and FIG. 9A.For these samples, <V_(mean)>_(rel) values exceeded 0.5, as shown inFIG. 9B.

On the other hand, pyrene nanoparticles obtained with the othersurface-altering agents, including PVA95K95, PVA13K98, PVA31K98, andPVA85K99, were predominantly or completely immobilized as demonstratedby respective <V_(mean)>_(rel) values of no greater than 0.5 and, withmost surface-altering agents, no greater than 0.4 (Table 11 and FIG.12B). Additionally, FIGS. 10A-10F are histograms showing distribution ofV_(mean) within an ensemble of particles. These histograms illustratemuco-diffusive behavior of samples stabilized with PVA2K75 and PVA9K80(similar histograms were obtained for samples stabilized with PVA13K87,PVA31K87, PVA85K87, and PVA130K87, but are not shown here) as opposed tomuco-adhesive behavior of samples stabilized with PVA31K98, PVA85K99,Kollidon 25, and Kollicoat IR (chosen as representative muco-adhesivesamples).

To identify the characteristics of the PVA that render pyrenenanoparticles mucus penetrating, <V_(mean)>_(rel) of the pyrenenanoparticles stabilized with various PVAs was mapped with respect to MWand hydrolysis degree of the PVAs used (FIG. 11). It was concluded thatat least those PVAs that have the hydrolysis degree of less than 95%rendered the nanoparticles mucus-penetrating.

Example 5

This example describes the measurement of the density of Pluronic® F127on the surface of particles comprising a nanoparticle core of apharmaceutical agent.

An aqueous dispersion containing a pharmaceutical agent and Pluronic®F127 was milled with milling media until particle size was reduced below300 nm. A small volume from the milled suspension was diluted to anappropriate concentration (˜100 μg/mL, for example) and the z-averagediameter was taken as a representative measurement of particle size. Theremaining suspension was then divided into two aliquots. Using HPLC, thefirst aliquot was assayed for the total concentration of drug (here,loteprednol eltabonate or fluticasone propionate) and for the totalconcentration of surface-altering moiety (here, Pluronic® F127). Againusing HPLC the second aliquot was assayed for the concentration of freeor unbound surface-altering moiety. In order to get only the free orunbound surface-altering moiety from the second aliquot, the particles,and therefore any bound surface-altering moiety, were removed byultracentrifugation. By subtracting the concentration of the unboundsurface-altering moiety from the total concentration of surface-alteringmoiety, the concentration of bound surface-altering moiety wasdetermined. Since the total concentration of drug was also determinedfrom the first aliquot, the mass ratio between the core material and thesurface-altering moiety can be determined. Using the molecular weight ofthe surface-altering moiety, the number of surface-altering moietymolecules to mass of core material can be calculated. To turn thisnumber into a surface density measurement, the surface area per mass ofcore material needs to be calculated. The volume of the particle isapproximated as that of a sphere with the diameter obtained from DLSallowing for the calculation of the surface area per mass of corematerial. In this way the number of surface-altering moieties persurface area is determined. FIG. 12 shows the results of surface-moietydensity determination for loteprednol etabonate and fluticasonepropionate.

Example 6. Formation of Mucus-Penetrating Particles Using Non-PolymericSolid Particles

The technique described in Example 1 was applied to other non-polymericsolid particles to show the versatility of the approach. F127 was usedas the surface-altering agent for coating a variety of activepharmaceuticals used as core particles. Sodium dodecyl sulfate (SDS) waschosen as a negative control so that each drug was compared to asimilarly sized nanoparticle of the same compound. An aqueous dispersioncontaining the pharmaceutical agent and Pluronic® F127 or SDS was milledwith milling media until particle size was reduced below 300 nm. Table12 lists the particle sizes for a representative selection of drugs thatwere milled using this method.

TABLE 12 Particle sizes for a representative selection of drugs milledin the presence of SDS and F127. Z-Ave D Drug Stabilizer (nm) PDIFluticasone F127 203 0.114 propionate SDS 202 0.193 Furosemide F127 2170.119 SDS 200 0.146 Itraconazole F127 155 0.158 SDS 168 0.163Prednisolone F127 273 0.090 SDS 245 0.120 Loteprednol F127 241 0.123etabonate SDS 241 0.130 Budesonide F127 173 0.112 SDS 194 0.135Indomethacin F127 225 0.123 SDS 216 0.154

In order to measure the ability of drug nanoparticles to penetrate mucusa new assay was developed which measures the mass transport ofnanoparticles into a mucus sample. Most drugs are not naturallyfluorescent and are therefore difficult to measure with particletracking microscopy techniques. The newly-developed bulk transport assaydoes not require the analyzed particles to be fluorescent or labeledwith dye. In this method, 20 μL of CVM is collected in a capillary tubeand one end is sealed with clay. The open end of the capillary tube isthen submerged in 20 μL of an aqueous suspension of particles which is0.5% w/v drug. After the desired time, typically 18 hours, the capillarytube is removed from the suspension and the outside is wiped clean. Thecapillary containing the mucus sample is placed in an ultracentrifugetube. Extraction media is added to the tube and incubated for 1 hourwhile mixing which removes the mucus from the capillary tube andextracts the drug from the mucus. The sample is then spun to removemucins and other non-soluble components. The amount of drug in theextracted sample can then be quantified using HPLC. The results of theseexperiments are in good agreement with those of the microscopy method,showing clear differentiation in transport between mucus penetratingparticles and conventional particles. The transport results for arepresentative selection of drugs are shown in FIG. 13. These resultscorroborate microscopy/particle tracking findings with pyrene anddemonstrate the extension to common active pharmaceutical compounds;coating non-polymeric solid nanoparticles with F127 enhances mucuspenetration.

Example 7. Synthesis of Hydrocortisone Derivatives

All compounds were synthesized as described below. The LC-MS method thatsupported the synthesis is as follows: column—Waters XTerra® MS C18, 3.5μm, 3.0×150 mm, column temperature—40° C., flow rate—0.6 mL/min,detection wavelength—254 nm, flow gradient—98:2 (0 minutes) to 0:100 (10minutes) 0.1% formic acid/H₂O:0.1% formic acid/acetonitrile.

Compound 2:17-[(2-Furanylcarbonyl)oxy]-11,21-dihydroxy-(11β)-pregn-4-ene-3,20-dione

Synthesis of 2-furancarboximidic acid methyl ester hydrochloride

Methanol (125 mL) was cooled in ice bath. Acetyl chloride (66.0 mL, 0.92mol) was slowly added. The solution was stirred while cooling in the icebath for 1 hour. The solution was stirred further for 1 hour at roomtemperature. Furan-2-carbonitrile (75.0 g, 0.81 mol) was added and thereaction was stirred overnight. Diethyl ether (500 mL) was added andthen suspension was stirred for 30 minutes. The precipitate wasfiltered. The solid was washed with additional diethyl ether (100 mL) toobtain product as an off-white solid. Yield 74.0 g, 56%.

Synthesis of 2-(trimethoxymethyl) furan

Potassium carbonate (300 g) was dissolved in water (1 L). The solutionwas cooled to room temperature and 2-furancarboximidic acid methyl esterhydrochloride (100.0 g, 0.62 mol) was added with stirring. The mixturewas extracted with ethyl acetate (2×500 mL). The organic solution wasdried with anhydrous magnesium sulfate and the solvent was evaporated.The residue was treated with hexanes (500 mL) and the solids werefiltered. The solvent was evaporated leaving colorless oil (62.0 g). Theoil was added to a solution of phosphoric acid (anhydrous, 48.2 g, 0.49mol) in dry methanol (700 mL). The solution was refluxed for 6 hours,then the precipitate was filtered and the solvent was evaporated.Hexanes (500 mL) was added to the residue and the precipitate wasfiltered. The solvent was evaporated to obtain the product as colorlessoil. Yield 55.0 g, 52%.

Synthesis of17-[(2-furanylcarbonyl)oxy]-11,21-dihydroxy-(11β)-pregn-4-ene-3,20-dione(Compound 1)

Hydrocortisone (25.00 g, 69.0 mmol), 2-(trimethoxymethyl)-furan (55.0 g,320.0 mmol) and pyridinium p-toluenesulfonate (6.5 g, 25.9 mmol) weredissolved in tetrahydrofuran (250 mL). The solution was heated to 70° C.for 3 hours. The solvent was evaporated. Dichloromethane (400 mL) wasadded followed by addition of hydrochloric acid (1.0 M, 200 mL). Themixture was vigorously stirred for 30 minutes. The organic phase wasseparated and dried with anhydrous magnesium sulfate. The solvent wasevaporated and the residue was dissolved in hexanes:dichloromethane 9:1(500 mL). The solution was applied on silica pad (300 g). The impuritieswere eluted with hexanes and the product mixture was eluted withdichloromethane:ethyl acetate 3:7. The solvent was evaporated leaving awhite solid (28.0 g) that consisted mostly of two isomers. The majorisomer was separated by flash chromatography (330 g silica column,hexanes to ethyl acetate). The fractions containing major isomer werecombined and concentrated to ca. 100 mL. The solution was sonicated toinduce formation of solid product, then the product was filtered anddried in high vacuum overnight to obtain a white solid. Yield: 13.0 g,41%. LC-MS: retention time 8.33 minutes, MS (positive ion) 345.2 (50%),457.3 (100%, M+1), 458.3 (30%, M+2), MS (negative ion) 491.1 (20%),501.2 (100%). ¹H NMR (CDCl₃) −7.61 (dd, J=2.0, 1.0 Hz, 1H), 7.19 (dd,J=3.5, 1.0 Hz, 1H), 6.53 (dd, J=4.0, 2.0 Hz, 1H), 5.71-5.70 (m, 1H),4.55-4.54 (m, 1H), 4.36 (dd, J=4.5, 23.0 Hz, 2H), 3.09 (br. s., 1H),2.95-2.85 (m, 1H), 2.56-2.44 (m, 2H), 2.42-2.34 (m, 1H), 2.30-2.18 (m,3H), 2.16-1.71 (m, 7H), 1.57-1.46 (m, 1H), 1.46 (m, 3H), 1.27-1.09 (m,3H), 0.99 (s, 3H).

Compound 3:17-[[2-(4-Bromophenyl)acetyl]oxy]-11,21-dihydroxy-(11β)-pregn-4-ene-3,20-dione

Synthesis of 4-bromobenzeneethanimidic acid methyl ester hydrochloride

4-Bromobenzeneacetonitrile (50.0 g, 0.26 mol) was dissolved in drymethanol (13 mL). Hexanes (125 mL) was added. The reaction mixture wascooled in ice bath and saturated with hydrogen chloride gas (generatedfrom 100 mL of concentrated hydrochloric acid slowly added to 250 mL ofconcentrated sulfuric acid). The ice bath was removed and the mixturewas stirred overnight. The solution was decanted from the formed semisolid. The semi-solid was suspended in diethyl ether (250 mL). Thesuspension was stirred for 30 minutes and the solid was filtered. Thesolid was washed with ether (100 mL). The solid was dried on the funnelby passage of vacuum for 30 minutes to obtain product as a white solid.Yield 69.0 g, 100%.

Synthesis of 1-bromo-4-(2,2,2-trimethoxyethyl)-benzene

4-Bromobenzeneethanimidic acid methyl ester hydrochloride (69.0 g, 0.26mol) was dissolved in dry methanol (80 mL). The solution was stirred for3 days. The precipitate was filtered and rinsed with diethyl ether (100mL). The solution was evaporated. Diethyl ether (250 mL) was added. Theprecipitate was filtered and the solvent was evaporated. Trace solventwas removed under high vacuum overnight to obtain product as a colorlessoil. Yield 63.7 g, 89%.

Synthesis of17-[[2-(4-bromophenyl)acetyl]oxy]-11,21-dihydroxy-(11β)-pregn-4-ene-3,20-dione(Compound 3)

Hydrocortisone (21.00 g, 58.0 mmol), (2,2,2-trimethoxyethyl)benzene(63.0 g, 229.1 mmol) and pyridinium p-toluenesulfonate (5.4 g, 21.5mmol) were dissolved in tetrahydrofuran (300 mL). The solution washeated to 70° C. for 3 hours. The solvent was evaporated. Diethyl ether(300 mL) was added and the precipitate was filtered, then dissolved indioxane (500 mL). Hydrochloric acid (1.0 M, 100 mL) was added. Themixture was vigorously stirred for 1 hour. The solution was diluted withwater (3.5 L) and stirred for 30 minutes. The precipitate was filteredand dissolved in dichloromethane (300 mL). The solution was dried withanhydrous magnesium sulfate, then solvent was evaporated to leave aresidue that that consisted mostly of two isomers. The mixture wasdissolved in ethyl acetate (500 mL). The solution was concentrated (ca.100 mL) and sonicated. The precipitate was filtered leaving behind awhite solid (17.6 g), which was further purified by flash chromatographyin dichloromethane to dichloromethane:ethyl acetate 1:1 (330 g silicacolumn). The fractions containing the more polar compound wereconcentrated to ca. 50 mL and hexanes was added until the solutionbecame cloudy. The suspension was sonicated to induce formation of solidproduct, which were filtered and dried in high vacuum overnight toobtain the product as a white solid. Yield: 12.91 g, 40%. LC-MS: LCretention time 9.22 minutes; MS (positive ion): 559.2 (100%, M+1), 560.2(30%, M+2), 561.2 (100%, M+1), 562.1 (30%, M+2), MS (negative ion):539.2 (100%), 540.2 (30%), 541.2 (100%), 542.2 (30%), 593.2 (15%), 595.2(15%). ¹H NMR (CDCl₃) −7.47-7.43 (m, 2H), 7.12-7.07 (m, 2H), 5.71 (br.s., 1H), 4.42 (br. s., 1H), 4.31-4.14 (2H), 3.58 (s, 2H), 3.03-3.01 (m,1H), 2.76-2.67 (m, 1H), 2.54-2.34 (m, 3H), 2.29-2.16 (m, 2H), 2.03-1.53(m, 10H), 1.41 (s, 3H), 1.11-0.95 (2H), 0.90 (s, 3H).

Compound 1:11,21-Dihydroxy-17-[1-oxo-3-(phenylsulfonyl)propoxy]-(11β)-pregn-4-ene-3,20-dione

Synthesis of 3-(phenylthio)propanenitrile

Thiophenol (40 mL, 0.39 mol) was dissolved in methanol (720 mL).Triethylamine was added (2.4 mL, 17.3 mmol). Acrylonitrile (23.2 mL,0.36 mol) was added dropwise over 1 hour. The solution was stirredovernight. The solvents were evaporated and the residual solvents wereremoved under high vacuum for 30 minutes at 70° C. to obtain thecompound as a yellow oil. Yield 59.4 g, 93%.

Synthesis of 3-(phenylthio)propanimidic acid ethyl ester hydrochloride

3-(Phenylthio)propanenitrile (59.4 g, 0.36 mol) was dissolved indichloromethane (300 ml) and ethanol (32 mL). The solution was cooled to−35° C. and saturated with hydrogen chloride gas (generated from 150 mLof concentrated hydrochloric acid slowly added to 300 mL of concentratedsulfuric acid over 1 hour. The cooling bath was removed and the mixturewas stirred overnight. The solvents were evaporated. The residualethanol was co-evaporated with dichloromethane (3×300 mL) to produceproduct as a thick colorless oil. Yield 97.4 g, 100%, containing ca. 10%residual solvent.

Synthesis of (3,3,3-triethoxypropyl)(phenyl)sulfane

3-(Phenylthio)propanimidic acid ethyl ester hydrochloride (97.4 g, 0.36mol) was dissolved in dichloromethane (300 mL) and dry ethanol (200 mL).The reaction mixture was stirred for 3 days. The precipitate wasfiltered. Diethyl ether (350 mL) was added and the precipitate wasfiltered. The addition of ether and filtration was repeated. Theresidual solvent was removed under high vacuum for 2 hours to obtain theproduct as a yellow oil. Yield 78.0 g, 76%.

Synthesis of11,21-Dihydroxy-17-[1-oxo-3-(phenylthio)propoxy]-(11β)-pregn-4-ene-3,20-dione

Hydrocortisone (20.00 g, 55.2 mmol),(3,3,3-triethoxypropyl)(phenyl)sulfane (32.0 g, 112.7 mmol) andpyridinium p-toluenesulfonate (5.2 g, 20.7 mmol) were dissolved intetrahydrofuran (300 mL). The solution was heated to 70° C. for 3 hours.The solvent was evaporated. Dichloromethane (300 mL) was added followedby addition of hydrochloric acid (1.0 M, 300 mL). The mixture wasvigorously stirred for 2 hours. The organic phase was separated, washedwith water (300 mL) and dried with anhydrous magnesium sulfate. Thesolvent was evaporated leaving an oily residue that consisted mostly oftwo isomers. The material was dissolved in dichloromethane (50 mL) andapplied in silica column (330 g), then separated by flash chromatographyusing dichloromethane to dichloromethane:ethyl acetate 1:1. Thefractions containing the more polar isomer were evaporated, leaving theproduct as a colorless oil. Yield 20.0 g, 69%, containing ca. 30%residual solvent. LC-MS: LC retention time 9.34 minutes, MS (positiveion) 527.3 (100%, M+1), 528.3 (30%), MS (negative ion): 461.2 (40%),561.2 (80%), 562.2 (25%), 571.3 (100%), 572.3 (30%).

Synthesis of11,21-dihydroxy-17-[1-oxo-3-(phenylsulfonyl)propoxy]-(11β)-pregn-4-ene-3,20-dione(Compound 2)

11,21-Dihydroxy-17-[1-oxo-3-(phenylthio)propoxy]-(11β)-pregn-4-ene-3,20-dione(20.0 g, 26.6 mmol) was dissolved in dichloromethane (500 mL). Thesolution was cooled in ice bath. mCPBA (20.0 g, 89.5 mmol, 77%) wasdissolved in dichloromethane (200 mL), then the mCPBA solution was addedover 1 hour. The solution was stirred for 1 hour, and then washed withaqueous sodium hydroxide (1.0 M, 2×300 mL) and water (300 mL). Thesolution was dried with anhydrous magnesium sulfate and the solvent wasconcentrated to 100 mL. The material was applied on silica column (330g) and purified by flash chromatography using dichloromethane to ethylacetate. The solvent was evaporated and dried in high vacuum overnightto obtain product as a white foam. Yield 12.5 g, 84%. LC-MS: LCretention time 8.28 minutes, MS (positive ion) 559.2 (100%, M+1), 560.3(30%), MS (negative ion) 557.3 (80%), 593.2 (25%), 603.3 (100%). ¹H NMR(CDCl₃) −7.93-7.88 (m, 2H), 7.72-7.24 (m, 1H), 7.62-7.55 (m, 2H), 5.70(br. s., 1H), 4.49 (br. s., 1H), 4.32 (q, J=18.5 Hz, 2H), 3.36 (td,J=7.5, 1.5 Hz, 2H), 3.02 (br. s., 1H), 2.78 (td, J=7.5, 1.5 Hz, 2H),2.75-2.68 (m, 1H), 2.55-2.33 (m, 3H), 2.30-2.15 (m, 2H), 2.11-1.98 (m,2H), 1.95-1.80 (m, 3H), 1.68-0.58 (4H), 1.52-1.44 (m, 1H), 1.44 (s, 3H),1.22-1.04 (m, 2H), 0.95 (s, 3H).

Example 8: Formulation of Compounds as Mucus-Penetrating Particles MediaMilling

All compounds were formulated using excipients and processes that canproduce MPPs. Specifically, the compounds were milled in the presence ofPluronic® F127 (F127) to 1) aid particle size reduction to severalhundreds of nanometers and 2) physically (non-covalently) coat thesurface of generated nanoparticles with a coating that would minimizeparticle interactions with mucus constituents and prevent mucusadhesion.

A milling procedure was employed in which aqueous dispersions containingcoarse compound particles were individually milled with F127 atnear-neutral pH buffer using a grinding medium. Briefly, a slurrycontaining 5% of compound and 5% F127 in PBS (0.0067 M PO₄ ³⁻), pH 7.1was added to an equal bulk volume of 1-mm ceria-stabilized zirconiumoxide beads in a glass vial (e.g., 2 mL of slurry per 2 mL of beads). Amagnetic stir bar was used to agitate the beads, stirring atapproximately 500 rpm at ambient conditions for 25 hours.

The milled suspensions were subjected to dynamic light scattering (DLS)measurements to determine particle size and polydispersity index (PDI, ameasure of the width of the particle size distribution). The samples forDLS measurements were buffered with HyClone™ PBS (Phosphate-BufferedSaline) to produce isotonic samples that have a physiologically relevantpH.

Table 13 summarizes the particle size and PDI of each compound aftermilling. The particle size and PDI of the milled suspensions ofcompounds 1 and 2 were reduced to <350 nm (z-averaged) and <0.20,respectively (Table 1). The purity of both compounds, as determined byhigh-performance liquid chromatography (HPLC), prior to millingwas >96%. After milling, purity remained >96. The purity of Compound 7after milling was <90%.

TABLE 13 Size (Z-averaged), PDI and chemical purity of milledsuspensions. Compound Size PDI Purity after milling 2 238 0.089 >96% 3341 0.197 >96% 1 509 0.318 <90%

The HPLC method used to determine the purity of milled suspensions is asfollows: column—SunFire™ C18, 3.5 μm, 3.0×150 mm, column temperature—40°C., flow rate—0.7 mL/min, detection wavelength—254 nm, flowgradient—50:50 (0 minutes) to 0:100 (10 minutes) 0.1% phosphoricacid/H₂O:acetonitrile.

Example 9. Crystalline Forms Sample Preparation, Procedure A for MilledSamples.

Particles were isolated by centrifugation, then resuspended in H₂O andthen recentrifuged. The wet sample was resuspended in H₂O and depositedthinly and evenly onto a flat zero background sample holder (Rigaku906165). The sample was allowed to air dry.

Sample Preparation, Procedure B for Neat Compound Samples.

Milligram amounts were packed as an evenly thin layer of solid onto azero background sample holder (Rigaku 906165).

Data Acquisition

XRPD patterns were obtained using a Rigaku MiniFlex 600 benchtop x-raydiffractometer equipped with a Cu X-ray tube (Cu/Kα=1.54059 Å), asix-position sample changer and a D/teX Ultra detector. XRPD patternswere acquired from 3-40° two theta at 0.02° step size and 5°/min scanspeed using the following instrument settings: 40 kV-15 mA X-raygenerator, 2.5° Soller Slit, 10 mm IHS, 0.625° Divergence Slit, 8 mmScatter Slit with Kβ filter, and an open Receiving Slit. Diffractionpatterns were viewed and analyzed using PDXL analysis software providedby the instrument manufacturer. A reference standard silicon powder(NIST Standard Reference Material 640d) generated a peak at 28.43° and28.45° two theta when samples were prepared as suspension in H₂O (tosimulate Procedure A) and Procedure B, respectively.

Samples

Except for Crystalline Form 1-B, the XRPD samples of all other formswere prepared using Procedure A (milled compounds) or Procedure B (neatcompounds). Procedure A was used to prepare the XRPD sample ofCrystalline Form 1-B.

Results

The crystal form summary of the input (before milling) and milledcrystal forms is shown in Table 14. All input crystalline forms werearbitrarily designated as “A” forms. New forms that emerged aftermilling, if any, were sequentially designated as “B”, “C”, etc. An “A”form was not designated for Compound 2 since the input material wasamorphous.

TABLE 14 Summary of Crystal Forms Before and After Milling. CrystalForms Crystal form change Input Final after milling Compound (beforemilling) (after milling) of Input forms? 2 2-A 2-A No 3 3-A 3-B Yes 1Amorphous 1-B Yes

Compound 2 did not change crystal form after milling while compounds 1and 3 changed forms after milling. Neat Form 3-B was prepared todemonstrate that it can be milled without undergoing further crystalform change. Neat form 1-B was not prepared due to chemical instabilityof compound during milling. Briefly, an aqueous suspension(approximately 400 mg in 4-6 mL H₂O) of 3-A was stirred at 40° C. for 1day to produce 3-B. The crystal form conversion experiment is describedin Table 15.

TABLE 15 Summary of neat crystal form conversion. Crystal Form InputFinal Stirring Stirring (before (after temperature time Compoundstirring) stirring) (° C.) (days) 3 3-A 3-B 40 1

Form 3-B was wet-milled using the same method that generated the data inTables 13 and 14. A comparison of milled particle size and PDI betweenthe input form 3-A and 3-B is shown in Table 16. Data shows that crystalform of the input “B” material was preserved during milling.

TABLE 16 Size (Z-average) and PDI of suspensions using differentstarting forms. Input Milled Suspension Compound Form* Final Form Size(nm) PDI 3 A B 341 0.197 B B 262 0.154 *Data for input 3-A form weretaken from Table 13.

A summary of the XRPD peaks is tabulated in Tables 17-20 and the XRPDpatterns of Forms 2-A, 3-A, 3-B and 1-B are shown in FIGS. 14-17.

TABLE 17 XRPD Peak Listing for Crystalline Form 2-A. Position ± 0.2d-spacing ± 0.2 Relative Intensity No. [°2θ] [Å] [%] 1 5.83 15.14 100.02 10.09 8.76 14.7 3 11.31 7.82 1.2 4 11.72 7.54 11.5 5 11.88 7.45 10.5 613.06 6.77 5.5 7 13.57 6.52 2.3 8 14.49 6.11 23.3 9 15.32 5.78 66.0 1015.66 5.65 24.9 11 16.72 5.30 12.4 12 17.61 5.03 7.7 13 17.98 4.93 10.014 18.43 4.81 3.5 15 19.65 4.51 1.4 16 20.35 4.36 7.5 17 20.51 4.33 1.918 21.00 4.23 9.9 19 21.36 4.16 4.5 20 22.09 4.02 4.6 21 22.75 3.91 16.122 23.16 3.84 3.9 23 23.72 3.75 5.2 24 25.10 3.54 2.4 25 25.88 3.44 3.126 27.40 3.25 0.7 27 28.45 3.14 1.2 28 28.63 3.11 0.8 29 29.62 3.01 2.930 30.43 2.94 1.1 31 33.16 2.70 1.1 32 34.74 2.58 1.6 33 35.77 2.51 0.434 36.33 2.47 0.4 35 36.80 2.44 0.2 36 37.42 2.40 1.4

TABLE 18 XRPD Peak Listing for Crystalline Form 3-A. Position ± 0.2d-spacing ± 0.2 Relative Intensity No. [°2θ] [Å] [%] 1 5.08 17.37 6.6 27.18 12.31 100.0 3 10.25 8.63 1.6 4 12.25 7.22 3.0 5 12.99 6.81 2.7 613.67 6.47 2.0 7 13.90 6.37 14.9 8 14.64 6.05 5.2 9 15.60 5.68 5.1 1016.87 5.25 4.7 11 18.18 4.88 3.2 12 18.73 4.73 6.3 13 19.63 4.52 2.9 1420.45 4.34 9.0 15 20.83 4.26 5.4 16 21.63 4.11 3.8 17 22.15 4.01 4.0 1824.00 3.71 5.3 19 25.58 3.48 2.8 20 26.16 3.40 0.6 21 27.04 3.30 3.3 2228.12 3.17 1.1 23 29.52 3.02 0.7 24 36.58 2.45 0.1

TABLE 19 XRPD Peak Listing for Crystalline Form 3-B. Position ± 0.2d-spacing ± 0.2 Relative Intensity No. [°2θ] [Å] [%] 1 5.25 16.81 4.1 28.49 10.41 4.1 3 8.88 9.95 36.7 4 10.55 8.38 19.6 5 11.01 8.03 1.9 611.91 7.43 13.6 7 12.66 6.98 100.0 8 14.34 6.17 80.3 9 14.57 6.08 17.410 15.05 5.88 24.8 11 15.66 5.65 4.3 12 15.97 5.55 15.3 13 16.58 5.349.2 14 17.88 4.96 14.5 15 19.02 4.66 97.8 16 19.64 4.52 10.3 17 20.284.37 59.0 18 20.63 4.30 59.8 19 22.14 4.01 9.0 20 22.30 3.98 13.3 2122.90 3.88 2.1 22 23.15 3.84 4.1 23 23.52 3.78 16.0 24 23.75 3.74 4.3 2524.20 3.67 2.9 26 24.78 3.59 3.4 27 25.01 3.56 1.2 28 25.47 3.49 6.2 2925.71 3.46 37.9 30 26.65 3.34 0.6 31 26.92 3.31 1.9 32 27.48 3.24 14.133 27.74 3.21 5.4 34 28.30 3.15 13.2 35 29.36 3.04 1.0 36 29.87 2.99 3.237 30.12 2.96 4.5 38 30.40 2.94 6.4 39 31.08 2.87 0.7 40 31.62 2.83 1.541 32.14 2.78 1.0 42 32.61 2.74 1.1 43 33.34 2.69 1.9 44 33.62 2.66 3.045 35.09 2.56 0.9 46 35.59 2.52 0.4 47 36.26 2.48 1.6 48 37.11 2.42 0.649 38.14 2.36 6.7 50 38.61 2.33 4.6

TABLE 20 XRPD Peak Listing for Crystalline Form 1-B. Position ± 0.2d-spacing ± 0.2 Relative Intensity No. [°2θ] [Å] [%] 1 5.88 15.03 12.2 210.36 8.53 57.1 3 13.18 6.71 53.4 4 13.40 6.60 28.8 5 14.40 6.15 10.7 615.12 5.86 2.5 7 15.55 5.69 100.0 8 15.91 5.57 2.4 9 17.57 5.04 20.8 1018.12 4.89 1.0 11 19.47 4.56 11.7 12 19.94 4.45 0.2 13 20.82 4.26 16.114 21.18 4.19 2.9 15 21.81 4.07 7.2 16 22.43 3.96 6.6 17 22.77 3.90 16.618 23.07 3.85 17.5 19 24.26 3.67 5.9 20 24.55 3.62 7.1 21 25.13 3.54 0.822 26.19 3.40 2.7 23 26.65 3.34 1.8 24 27.07 3.29 3.5 25 27.39 3.25 0.626 27.83 3.20 0.3 27 28.28 3.15 1.4 28 28.67 3.11 1.3 29 29.05 3.07 0.530 30.10 2.97 0.1 31 30.54 2.92 1.3 32 31.87 2.81 0.1 33 32.26 2.77 1.334 32.92 2.72 1.4 35 33.49 2.67 0.2 36 34.04 2.63 0.4 37 34.61 2.59 0.538 34.92 2.57 1.1 39 35.15 2.55 0.9 40 35.72 2.51 0.3 41 36.21 2.48 0.442 36.69 2.45 0.5 43 37.57 2.39 1.2 44 38.02 2.36 0.1 45 38.43 2.34 0.846 38.67 2.33 0.2 47 39.52 2.28 0.3 48 39.76 2.27 0.9

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used hereinthe terms “about” and “approximately” means within 10 to 15%, preferablywithin 5 to 10%. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof the invention are approximations, the numerical values set forth inthe specific examples are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in their respective testingmeasurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. A pharmaceutical composition, comprising: a plurality of coated particles, each coated particle comprising: a core particle comprising a hydrocortisone derivative selected from

and a mucus penetration-enhancing coating surrounding the core particle, wherein the mucus penetration-enhancing coating comprises a surface-altering agent comprising one or more of the following components: a) a triblock copolymer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration, wherein the hydrophobic block has a molecular weight of at least about 2 kDa, and the hydrophilic blocks constitute at least about 15 wt % of the triblock copolymer, wherein the hydrophobic block associates with the surface of the core particle, and wherein the hydrophilic block is present at the surface of the coated particle and renders the coated particle hydrophilic, b) a synthetic polymer having pendant hydroxyl groups on the backbone of the polymer, the polymer having a molecular weight of at least about 1 kDa and less than or equal to about 1000 kDa, wherein the polymer is at least about 30% hydrolyzed and less than about 95% hydrolyzed, or c) a polysorbate, wherein the surface altering agent is present on the outer surface of the core particle at a density of at least 0.01 molecules/nm², wherein the surface altering agent is present in the pharmaceutical composition in an amount of between about 0.001% to about 5% by weight; and one or more pharmaceutically acceptable carriers, additives, or diluents; wherein the pharmaceutical composition is suitable for administration to an eye of a subject.
 2. The pharmaceutical composition of claim 1, wherein the coating on the core particle is present in a sufficient amount to increase the concentration of the hydrocortisone derivative in an ocular tissue after administration when administered to the eye, compared to the concentration of the hydrocortisone derivative in the ocular tissue when administered as a core particle without the coating. 3.-5. (canceled)
 6. The pharmaceutical composition of claim 1, wherein the hydrocortisone derivative is


7. The pharmaceutical composition of claim 6, wherein Compound 1 is in crystalline form B having X-ray powder diffraction (XRPD) peaks at 5.88, 10.36, 13.18, 14.40, 15.55, 17.57, and 20.82±0.2°2θ.
 8. The pharmaceutical composition of claim 1, wherein the hydrocortisone derivative is


9. The pharmaceutical composition of claim 8, wherein Compound 2 is in crystalline form A having XRPD peaks at 5.83, 10.09, 11.72, 14.49, 15.32, and 15.66±0.2° 2θ.
 10. The pharmaceutical composition of claim 1, wherein the hydrocortisone derivative is


11. The pharmaceutical composition of claim 10, wherein Compound 3 is in crystalline form A having XRPD peaks at 5.08, 7.18, 13.90, and 20.45±0.2° 2θ.
 12. The pharmaceutical composition of claim 10, wherein Compound 3 is in crystalline form B having XRPD peaks at 8.88, 12.66, 14.34, 19.02, 20.28, 20.63 and 25.71±0.2° 2θ.
 13. The pharmaceutical composition of claim 1, wherein the surface-altering agent is present on the surfaces of the coated particles at a density of at least about 0.1 molecules per nanometer squared.
 14. The pharmaceutical composition of claim 1, wherein the surface-altering agent is covalently attached to the core particles.
 15. The pharmaceutical composition of claim 1, wherein the surface-altering agent is non-covalently adsorbed to the core particles.
 16. The pharmaceutical composition of claim 1, wherein the surface-altering agent comprises the triblock copolymer.
 17. The pharmaceutical composition of claim 16, wherein the hydrophilic blocks of the triblock copolymer constitute at least about 30 wt % of the triblock polymer and less than or equal to about 80 wt % of the triblock copolymer.
 18. The pharmaceutical composition of claim 17, wherein the hydrophobic block portion of the triblock copolymer has a molecular weight of about 3 kDa to about 8 kDa.
 19. The pharmaceutical composition of claim 17, wherein the triblock copolymer is poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide).
 20. The pharmaceutical composition of claim 1, wherein the surface-altering agent has a molecular weight of at least about 4 kDa.
 21. The pharmaceutical composition of claim 1, wherein the surface-altering agent comprises a linear polymer having pendant hydroxyl groups on the backbone of the polymer.
 22. The pharmaceutical composition of claim 21, wherein the surface altering agent is poly(vinyl alcohol).
 23. The pharmaceutical composition of claim 22, wherein the poly(vinyl alcohol) is about 70% to about 94% hydrolyzed.
 24. The pharmaceutical composition of claim 1, wherein the hydrocortisone derivative is crystalline.
 25. The pharmaceutical composition of claim 1, wherein the hydrocortisone derivative is amorphous.
 26. The pharmaceutical composition of claim 1, wherein the hydrocortisone derivative is encapsulated in a polymer, a lipid, a protein, or a combination thereof.
 27. The pharmaceutical composition of claim 1, wherein the hydrocortisone derivative comprises at least about 80 wt % of the core particle.
 28. The pharmaceutical composition of claim 1, wherein the coated particles have an average size of about 10 nm to about 1 μm.
 29. The pharmaceutical composition of claim 1, comprising one or more degradants of the hydrocortisone derivative, and wherein the concentration of each degradant is 0.1 wt % or less relative to the weight of the hydrocortisone derivative.
 30. The pharmaceutical composition of claim 1, wherein the polydispersity index of the composition is less than or equal to about 0.5.
 31. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is suitable for topical administration to the eye.
 32. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is suitable for direct injection into the eye.
 33. The pharmaceutical composition of any claim 1, wherein the one or more ophthalmically acceptable carriers, additives, or diluents comprises glycerin.
 34. A method of treating, diagnosing, preventing, or managing an ocular condition in a subject, the method comprising: administering a pharmaceutical composition of claim 1 to an eye of a subject and thereby delivering the hydrocortisone derivative to a tissue in the eye of the subject.
 35. The method of claim 34, comprising sustaining an ophthalmically efficacious level of the hydrocortisone derivative and/or its hydrocortisone metabolite in a palpebral conjunctiva, a fornix conjunctiva, a bulbar conjunctiva, or a cornea for at least 12 hours after administration.
 36. The method of claim 34, comprising delivering the hydrocortisone derivative and/or its hydrocortisone metabolite to a tissue in the front of the eye of the subject.
 37. The method of claim 34, comprising delivering the hydrocortisone derivative and/or its hydrocortisone metabolite to a tissue in the back of the eye of the subject.
 38. The method of claim 37, wherein the tissue is a retina, a macula, a sclera, a cornea, a lid, aqueous humor, or a choroid.
 39. The method of claim 34, wherein the ocular condition is inflammation, macular degeneration, macular edema, uveitis, glaucoma, or dry eye.
 40. The pharmaceutical composition of claim 28, wherein the average particle size is measured by dynamic light scattering.
 41. The pharmaceutical composition of claim 30, wherein the polydispersity index is measured by dynamic light scattering.
 42. The pharmaceutical composition of claim 2, wherein the ocular tissue is an anterior ocular tissue.
 43. The pharmaceutical composition of claim 42, wherein the anterior ocular tissue is a palpebral conjunctiva, a bulbar conjunctiva, a fornix conjunctiva, an aqueous humor, an anterior sclera, a cornea, an iris, or a ciliary body.
 44. The pharmaceutical composition of claim 2, wherein the ocular tissue is a tissue at the back of the eye.
 45. The pharmaceutical composition of claim 42, wherein the anterior ocular tissue is a vitreous humor, a vitreous chamber, a retina, a macula, a choroid, a posterior sclera, a uvea, an optic nerve, or the blood vessels or nerves which vascularize or innervate a posterior ocular region or site of they eye. 